Rapid detection of antibiotic resistance in mycobacterium tuberculosis

ABSTRACT

PCT No. PCT/EP93/01063 Sec. 371 Date May 9, 1995 Sec. 102(e) Date May 9, 1995 PCT Filed Apr. 30, 1993 PCT Pub. No. WO93/22454 PCT Pub. Date Nov. 11, 1993A process for the detection of resistance to an antibiotic in a mycobacterium comprises detecting a mutation in a gene selected from the group consisting of the katG gene or fragment thereof, the rpoB gene or fragment thereof, and the rpsI gene or fragment thereof. The process is useful for detecting in vitro the presence of nucleic acids of a Mycobacterium tuberculosis resistant to isoniazid.

This application is a National stage application filed under 35 U.S.C. §371 based on International Application PCT/EP/01063, filed Apr. 30,1993, which is based upon U.S. application Ser. No. 07/929,206, filedAug. 14, 1992, now U.S. Pat. No. 5,633,131, issued May 27, 1997, whichis a continuation-in-part application of U.S. application Ser. No.07/875,940, filed Apr. 30, 1992, now abandoned and French applicationsNo. FR 93 04545, filed Apr. 16, 1993, and No. FR 92 11098, filed Sep.17, 1992.

This application is a National stage application filed under 35 U.S.C. §371 based on International Application PCT/EP/01063, filed Apr. 30,1993, which is based upon U.S. application Ser. No. 07/929,206, filedAug. 14, 1992, now U.S. Pat. No. 5,633,131, issued May 27, 1997, whichis a continuation-in-part application of U.S. application Ser. No.07/875,940, filed Apr. 30, 1992, now abandoned and French applicationsNo. FR 93 04545, filed Apr. 16, 1993, and No. FR 92 11098, filed Sep.17, 1992.

This invention relates to the rapid detection of strains ofMycobacterium tuberculosis that are resistant to antibiotics,particularly isoniazid, rifampicin and streptomycin. More particularly,this invention relates to a method of detecting antibiotic resistance inMycobacterium tuberculosis, e.g. either as a result of mutations in therelevant genes or by nucleic acid hybridization. This invention alsorelates to a nucleic acid probe and a kit for carrying out the nucleicacid hybridization. The invention further relates to the chromosomallocation of the katG gene (SEQ ID NO:45) and its nucleotide sequence.

BACKGROUND OF THE INVENTION

Despite more than a century of research since the discovery ofMycobacterium tuberculosis, the aetiological agent of tuberculosis, byRobert Koch, this disease remains one of the major causes of humanmorbidity and mortality. There are an estimated 3 million deathsannually attributable to tuberculosis (Snider, 1989), and although themajority of these are in developing countries, the disease is assumingrenewed importance in the West due to the increasing number of homelesspeople and the impact of the AIDS epidemic (Chaisson et al., 1987;Snider and Roper, 1992).

Isonicotinic acid hydrazide or isoniazid (INH) has been used in thetreatment of tuberculosis for the last forty years due to its exquisitepotency against the members of the "tuberculosis" groups--Mycobacteriumtuberculosis, M. bovis and M. africanum (Middlebrook, 1952; Youatt,1969). Neither the precise target of the drug, nor its mode of action,are known, and INH treatment results in the perturbation of severalmetabolic pathways. There is substantial evidence indicating that INHmay act as an antimetabolite of NAD and pyridoxal phosphate (Bekierkunstand Bricker, 1967; Sriprakash and Ramakrishnan, 1970; Winder andCollins, 1968, 1969, 1970), and other data indicating that the drugblocks the synthesis of the mycolic acids, which are responsible for theacid-fast character of mycobacterial cell walls (Winder and Collins1970; Quemard et al., 1991). Shortly after its introduction,INH-resistant isolates of Mycobacterium tuberculosis emerged and, oncharacterization, were often found to have lost catalase-peroxidaseactivity and to show reduced virulence in guinea pigs (Middlebrook etal., 1954; Kubica et al., 1968; Sriprakash and Ramakrishnan, 1970).

Very recently, INH-resistance has acquired new significance owing to atuberculosis epidemic in the USA due to multidrug resistant (MDR)variants of M. tuberculosis (CDC, 1990; 1991a, b) and the demonstrationthat such strains were responsible for extensive nosocomial infectionsof HIV-infected individuals and health care workers (Snider and Roper,1992). In view of the gravity of this problem, there exists a need inthe art to determine the relationship between INH-resistance andcatalase-peroxidase production.

More particularly, there is a need in the art to understand themolecular mechanisms involved in drug sensitivity. In addition, there isa need in the art to develop a simple test permitting the rapididentification of INH-resistant strains. Further, there is a need in theart for reagents to carry out such a test.

Rifampicin too is a major antibiotic used for the treatment ofinfections by mycobacterium, particularly Mycobacterium tuberculosis andMycobacterium leprae. Because some mycobacteria grow slowly, possiblerapid and efficient tests for the testing of resistance to rifampicin oranalogues thereof must be made available. Likewise the invention aims ata rapid detection of strands of Mycobacterium tuberculosis which areresistant to streptomycin. Because of the development of resistance tostreptomycin, the latter antibiotic has been used together with otherantibiotics, e.g. isoniazid. Thus adequat treatment of tuberculosisshould be preceded by rapid and efficient detection of resistances tothe three major antibiotics, isoniazid, rifampicin and streptomycin.

SUMMARY OF THE INVENTION

Accordingly, this invention aids in fulfilling these needs in the art byproviding a process for detecting in vitro the presence of cells of aMycobacterium tuberculosis resistant to isoniazid and other drugs, suchas rifampicin or analogues thereof, and streptomycin.

By analogues of rifampicin, a particularly meant derivatives of3-formyl-rifamycin, particularly as a result of substitution therein forthe substituent present either in the naphtofuranonyl group or of thesite chain at position 7 of the naphtofuranonyl group, or by theintroduction or removal of a double band in the lateral chain.

In accordance with the invention, the detection of a resistance toisoniazid involves the detection of one or several ligations in the katGgene (SEQ ID NO:45) of Mycobacterium tuberculosis, particularly withrespect to the nucleotide sequence of that same katG gene (SEQ ID NO:45)in mycobacterium tuberculosis that are not resistant to isoniazid.

Another process alternative for detecting in vitro the presence ofnucleic acids of a Mycobacterium tuberculosis resistant to isoniazid,wherein the process comprises the steps of:

contacting said nucleic acids previously made accessible to a probe ifrequired under conditions permitting hybridization;

detecting any probe that had hybridized to said nucleic acids;

wherein said probe comprises a nucleic acid sequence, which is 2.5 kbEcoRV-KpnI fragment of plasmid pYZ56 or of part thereof, and whereinsaid fragment contains a BamHI cleavage site, wherein said part isnonetheless sufficiently long to provide for the selectivity of the invitro detection of a Mycobacterium tuberculosis resistant to isoniazid.

For instance, this process alternative comprises the steps of:

(A) depositing and fixing nucleic acids of the cells on a solid support,so as to make the nucleic acids accessible to a probe;

(B) contacting the fixed nucleic acids from step (A) with a probe underconditions permitting hybridization;

(C) washing the filter resulting from step (B), so as to eliminate anynon-hybridized probe; and then

(D) detecting any hybridized probe on the washed filter resulting fromstep (C).

The probe comprises a nucleic acid sequence which is present in a 2.5 kbEcoRV-KpnI fragment of plasmid pYZ56, wherein said fragment contains aBamHI cleavage site. This fragment has been found to be associated withintracellular DNA of isoniazid-sensitive Mycobacterium tuberculosis andis capable of distinguishing such antibiotic sensitive microorganismsfrom isoniazid-resistant Mycobacterium tuberculosis, which do notcontain DNA that hybridizes with this fragment under the conditionsdescribed hereinafter.

This invention further provides nucleotide sequences, such as RNA andDNA, of isoniazid-resistant Mycobacterium tuberculosis encoding theregion of the katG gene (SEQ ID NO:45) of Mycobacterium tuberculosisthat imparts isoniazid sensitivity absent from isoniazid-resistantcells.

This invention also provides a probe consisting of a label, such as aradionucleotide, bonded to a nucleotide sequence of the invention.

In addition, this invention provides a hybrid duplex molecule consistingessentially of a nucleotide sequence of the invention hydrogen bonded toa nucleotide sequence of complementary base sequence, such as DNA orRNA.

Also, this invention provides a process for selecting a nucleotidesequence coding for a catalase-peroxidase gene of Mycobacteriumtuberculosis, or for a portion of such a nucleotide sequence, from agroup of nucleotide sequences, which comprises the step of determiningwhich of the nucleotide sequences hybridizes to a nucleotide sequence ofthe invention. The nucleotide sequence can be a DNA sequence or an RNAsequence. The process can include the step of detecting a label on thenucleotide sequence.

Further, this invention provides a kit for the detection ofMycobacterium tuberculosis resistant to isoniazid. The kit comprises acontainer means containing a probe comprising a nucleic acid sequence,which is a 2.5 kb EcoRV-KpnI fragment of plasmid pYZ56, wherein thefragment contains a BamHI cleavage site. The kit also includes acontainer means containing a control preparation of nucleic acid.

The invention also covers compounds obtained as products of the actionof the enzyme catalase, or a similar enzyme on isoniazid. The katG gene(SEQ ID NO:45) or a derivative of this gene which retains a similaractivity can be used as a source of catalase protein. The new compoundsare selected by reactivity on INH-resistant-mycobacterial strains by theantibiogram method such as described in H. David et al. 's "Methodes delaboratoire pour Mycobacteriologie clinique" edited by PasteurInstitute, ISBN N 0995-2454.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be described in greater detail by reference to thedrawings in which:

FIG. 1. shows the INH-resistant M. smegmatis strain, BH1 (Gayathri etal., 1975) (a derivative of strain MC² -155) was transformed with a poolof M. tuberculosis-H37Rv shuttle cosmids (kindly provided by Dr. W. R.Jacobs, New York) and individual clones were scored forINH-susceptibility. Cosmid pBH4 consistently conferred drugsusceptibility and the transformant overproduced catalase (assayed as inHeym, 1992). The restriction map of the DNA insert from pBH4 is shownalong with that of the insert from pYZ55 --a plasmid containing katG ofM. tuberculosis H37Rv, isolated on the basis of hybridization with anoligonucleotide probe (5'-TTCATCCGCATGGCCTGGCACGGCGCGGGCACCTACCGC-3')(SEQ ID NO:1) designed to match the amino acid sequence from a conservedregion of E. coli hydroperoxidase I (HPI). Restriction sites for thefollowing enzymes are indicated : B, BamHI;C, ClaI; E, EcoRV; H,HindIII, K, KpnI; M, SmaI; N, NotI; R, EcoRI; S, SacI.

Transformation of BHl with a mycobacterial shuttle plasmid, pBAK14,Zhang et al., 1991, containing the 4.5 kb insert from pYZ55 similarlyconferred INH-susceptibility. MIC's are also shown for BHI transformedwith subfragments derived from pYZ55 and inserted into pBAK14 in one (+)or other (-) orientation. The katG gene (SEQ ID NO:45) and the abilityto confer INH-susceptibility both mapped to a 2.9 kb EcoRV-KpnI fragment(pBAK-KE+).

FIG. 2 shows extracts from M. tuberculosis H37Rv and from E. colistrains transformed with a variety of plasmid constructs that wereprepared for activity gel analysis as described previously (Zhang etal., 1991). Non-denaturing gels containing 8% polyacrylamide werestained for catalase (panel A) and peroxidase (panel B) activities asdescribed by Wayne and Diaz (Wayne et al., 1986). Lane 1, M.tuberculosis H37Rv; 2, E. coli UM2 (katE, katG; 3, E. coli UM2/pYZ55; 4,E. coli UM2/pYZ56 (the 2.9 kb EcoRV-Kpn1 fragment in pUC19,corresponding to pBAK-KE+in FIG. 1); 5, E. coli UM2/pYZ57 (pYZ55 with aBamHl-Kpnl deletion, corresponding to pBAK-KB+ in FIG. 1). M.tuberculosis catalase and peroxidase activities migrated as two bandsunder these conditions (lane 1); the same pattern was seen for therecombinant enzyme expressed by pYZ55 (lane 3). pYZ56 (lane 4) expressesa protein of increased molecular weight due to a fusion between katG andlacZ' from the vector as shown in panel C. Panel C also shows partialsequence alignment with E. coli HPI (SEQ ID NOS:42-44).

FIG. 3 shows an E coli strain with mutations in both katG and KatE (UM2Mulvey et al., 1988) that was transformed with pUC19 vector alone, pYZ55expressing M. tuberculosis katG and pYZ56 with high level expression ofM. tuberculosis katG. Overnight cultures in Luria-Bertani brothsupplemented with appropriate antibiotics were plated out in thepresence of varying concentrations of INH and colony forming units wereassessed. Results of a representative experiment are shown with errorbars indicating the standard deviation observed in triplicate samples.Overexpression of M. tuberculosis katG similarly conferredsusceptibility to high concentrations of INH in E. coli UM255 (katG,katE, Mulvey et al., 1988), but had no effect on catalase-positivestrains such as E. coli TG1. In some experiments, high concentrations ofINH had detectable inhibitory effect on growth of UM2 and UM255, alone,but in all experiments inhibition of pYZ56-transformants was at least10-100 fold greater than that observed in the corresponding vectorcontrols.

FIG. 4 shows Southern blots prepared using genomic DNA from different N.tuberculosis strains, digested with Kpn1, that were probed with (A) katG(the 4.5 kb Kpn1 fragment)(SEQ ID NO:45), and (B) the SOD gene (1.1 kbEcoR1-Kpn1 fragment, Zhang et al., 1991). Labelling of probes andprocessing of blots was performed as described previously (Eiglmeier etal., 1991; Maniatis et al., 1989). Lane 1, H37Rv; 2, strain 12 -MIC 1.6μg/ml INH; 3, Bl453 -MIC>50 μg/ml INH (Jackett et al., 1978); 4, strain24 -MIC>50 μg/m1 INH; 5, 79112 -INH-sensitive (Mitchison et al., 1963);6, 12646 -INH-sensitive (Mitchison et al., 1963); 7, 79665-INH-sensitive (Mitchinson et al., 1963). INH susceptibilities wereconfirmed by inoculation of Lowenstein-Jensen slopes containingdiffering concentrations of INH.

FIG. 5. Organization of the katG locus. The upper bar corresponds to astretch of the M. tuberculosis chromosome spanning the katG region andthe positions of individual cosmids used to construct the map are shownbelow together with the original shuttle cosmid pBH4 and pYZ55. Thelocations of some key restriction sites (B, BamHI; K, KpnI) are showntogether with the approximate location of the known genetic markers:fbpB encoding the alpha or 85-B antigen (Matsuo et al., 1988); katG,catalase-peorxidase; LL105, an anonymous λgtll clone kindly supplied byA Andersen; MPTR, major polymorphic tandem repeat (Hermans et al.,1992).

FIG. 6. A. Nucleotide sequence of the KpnI fragment bearing katG (SEQ IDNO:45) . This sequence has been deposited in the EMBL data-library underaccession number X68081. The deduced protein sequence is shown in theone letter code.

B. Alignment of the two copies of the 700 bp direct repeat withidentities shown as * and --denoting pads introduced to optimize thealignment (SEQ ID NOS: 46-47). Numbering refers to the positions in FIG.2A.

FIG. 7. Distribution of katG in mycobacteria. A. Samples of differentbacterial DNAs (1.5 ug) were digested with RsrII, separated by agarosegel electrophoresis and stained with ethidium bromide; lanes 1 and 7,size markers; M. leprae; lane 3, M. tuberculosis H37Rv; lane 4, M.gordonae; lane 5, N. szulgai; lane 6, M. avium. B. Hybridization of thegel in A, after Southern blotting, with a katG specific probe.

FIGS. 8(1) and 8(2). Primary structure alignment of catalase-peroxidases(SEQ ID NOS:48-53). The sequences are from M. tuberculosis H37RV, mtkatg(SEQ ID NO:48); E. coli, eckatg (SEQ ID NO:49)(Triggs-Raine et al.,1988); S. typhimurium, stkatg (SEQ ID NO:50); B. stearothermophilus,bspera (SEQ ID NO:51)(Loprasert et al., 1988) and yeast cytochrome cperoxidase (SEQ ID NO:52) (ccp; Finzel et al., 1984). The alignment wasgenerated using PILEUP and PRETTY (Devereux et al., 1984) and . denotegaps introduced to maximize the homology. Key residues from the activesite and the peroxidase motifs (Welinder, 1991), discussed in the text,are indicated below the consensus.

FIG. 9. Western blot analysis of M. tuberculosis KatG (SEQ ID NO:45)produced in different bacteria. Proteins were separated bySDS-polyacrylamide gel electrophoresis then subjected to immunoblotting,and detection with antiserum raised against BCG, as described in Zhanget al., 1991.

Lane 1, soluble extract of M. tuberculosis H37Rv; lane 2, M. smegmatisMC² 155 harboring the vector pBAK14; lane 3, M 155 harboring pBAK-KK(katG+); lane 4, E. coli UM2 (katE, katG), lane 5, UM2 harboring pYZ55(katG⁺); lane 6, UM2 harboring pYZ56 (lacZ'::katG), having regard to thedrawings in which:

FIG. 10 represents diagrammatically the PCR strategy used for the studyof different M. Leprae isolates, showing the coding sequence of rpoBsequence, whereby the sequenced regions are shown by hatched parts, andthe position and reference of the amplification primers used beingindicated on the upper line, whereas the sequencing primers areindicated bellow it;

FIG. 11 represents (A) the nucleotidic sequence of a short region ofrpoB (SEQ ID NO:54) carrying the mutations that confer resistance torifampicin with an indication of the changes of bases in thecorresponding alleles and (B) a comparison between the amino acidssequences of the domain I of region II of the β-sub-unit of the RNApolymerase of E. coli (SEQ ID NO:55) and M. Leprae (SEQ ID NO:56),whereby the numbers of the residues and the differences in the mutatedaminoacids have been indicated; the mutated amino acid residuesassociated with rifampicin resistance as well as the frequency of itsoccurrences have been represented too,

FIG. 12 shows a complete sequence of the rpoB gene of M. Leprae (SEQ IDNO:56),

FIG. 13 represents the sequence of part of the rpoB gene of M.tuberculosis (SEQ ID NOS:59-60),

FIG. 14 represents the sequence of a part of the rpsL gene of M.tuberculosis (SEQ ID NOS:63-64); both the sequence of the full rpsL geneof M. Leprae and that of its expression product (SEQ ID NOS:61-62), thatis the S12 protein (whose starting aminoacid is noted by 1) areindicated. The positions of the ML51 (SEQ ID NO:40) and ML52 (SEQ IDNO:41) primers, as well as of the sequences of part of the rpsL gene ofM. tuberculosis are provided below those of M. Leprae. Only thosepositions which are different and the corresponding amino acid changesare indicated.

FIG. 15 represents the wild DNA sequence of the rpsL gene (SEQ ID NO:65)fragment coding for the S12 protein of the small ribosome sub-unit thatis responsible for the resistance to streptomycin, as well as thecorresponding aminoacid sequence of the S12 protein (SEQ ID NO:66).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

The recent emergence of large numbers of strains of M. tuberculosisshowing multidrug resistance in the United States is a most alarmingdevelopment given the extreme contagiousness of this organism. Thisdanger has been strikingly illustrated by several small tuberculosisepidemics in which a single patient infected with MDR M. tuberculosishas infected both HIV-positive individuals, prison guards and healthynursing staff (CDC 1990, 1991; Daley et al., 1992; Snider and Roper,1992). Given the gravity of the current worldwide HIV epidemic, it isconceivable that if AIDS patients in the West, like those in Africa,were to be infected with MDR M. tuberculosis strains (rather thanmembers of the M. avium/M. intracellulare complex) widespreaddissemination of the disease would result.

Isoniazid (INH) is a bactericidal drug which is particularly potentagainst the tuberculosis group of mycobacteria--Mycobacteriumtuberculosis, M. bovis, and M. africanum--and, in consequence, it hasbeen particularly effective in the treatment of tuberculosis. Standardanti-tuberculosis regimens generally include INH and rifampicin, oftenin combination with the weaker drugs, pyrazinamide, ethambutol orstreptomycin. Besides its use in therapy INH is also given to closecontacts of patients as a prophylactic measure.

INH-resistant mutants of M. tuberculosis, the agent of the humandisease, show two levels of resistance: low (1 to 10 μg/ml) and high (10to 100 μg/ml). INH-resistance is often associated with loss of catalaseactivity and virulence. Recently, owing to the AIDS epidemic, increasedhomelessness and declining social conditions, tuberculosis has reemergedas a major public health problem in developed countries, particularlythe USA. An alarming feature of the disease today is the emergence ofmultiple drug-resistant organisms and rapid nosocomial transmission tohealth care workers and HIV-infected patients. This has prompted CDC topropose new recommendations for the treatment of multiple resistantstrains (at least INH and rifampicin) and the prevention oftransmission. To obtain fresh insight into the problem of INH-resistanceand to develop a rapid diagnostic test the following study wasperformed.

Clearly, it is essential to understand the mechanisms of resistance toINH and rifampicin, the main anti-tuberculosis agents, as this willallow novel chemotherapeutic strategies to be developed and facilitatethe design of new compounds active against MDR strains.

This invention demonstrates that it is the catalase-peroxidase enzyme,HPI, which is the INH target, and it is suggested that this enzyme alonemediates toxicity. Compelling evidence of this conclusion was obtainedby expression of the M. tuberculosis katG gene (SEQ ID NO:45) in acatalase-negative mutant of E. coli as this resulted in this bacteriumbecoming sensitive to INH. Moreover, the isolation of the M.tuberculosis INH-sensitivity gene, katG (SEQ ID NO:45), is important asit will facilitate the rapid detection of INH-resistant strains by meansof hybridization and PCR-based approaches. The high frequency ofdeletions in clinical strains, as shown here, should simplify thisprocedure.

Identification of an M. tuberculosis gene involved in INH-sensitivity

A heterologous approach was employed to isolate M. tuberculosis gene(s)involved in INH-sensitivity. BH1 is a spontaneous mutant of the easilytransformable M. smegatis strain MC² 155 (Snapper et al., 1990), that isresistant to 512 μg/ml of the INH and lacks catalase-peroxidase activity(Heym et al., 1992). As there is a strict correlation betweenINH-sensitivity and these enzyme activities, transformation of BH1 witha plasmid carrying the appropriate gene from M. tuberculosis should leadto their restoration and concomitant INH-sensitivity.

Consequently, DNA was prepared from a pool of M. tuberculosis shuttlecosmids in Escherichia coli and introduced into BN1 byelectro-transformation. Over 1000 kanamycin-resistant transformants werethen scored for INH-sensitivity, and four clones that failed to grow onmedium containing 32 g/ml of INH, the MIC from wild type strain M² 155,were obtained.

After re-transformation of BH1, only one of these, pBN4, consistentlyconferred the INH-sensitive phenotype. Restriction digests with BamHI,KpnI, NotI, ClaI and HindIII showed the M. tuberculosis chromosomal DNAcarried by pBH4 to be about 30 kb in size. A map produced with the lastthree enzymes is presented in FIG. 1.

When pBN4 was used as a hybridization probe to detect homologous clonesin the library, a further eight shuttle cosmids were isolated. Ontransformation into BH1, five of these (T35, T646, T673, T79, T556)restored INH-sensitivity, and showed similar restriction profiles topBN4. In particular, a KpnI fragment of 4.5 kb was present in all cases.

Attempts to subclone individual BamHI fragments did not give rise totransformants capable of complementing the lesion in BH1 suggesting thata BamHI site might be located in the gene of interest. In contrast,pBH5, a derivative of pBH4, was constructed by deletion of EcoRIfragments and this showed that a 7 kb segment was not required forrestoration of INH-sensitivity.

Transformants harboring shuttle cosmids that complemented theINH-resistant mutation of BH1 were examined carefully and the MICs forseveral antibiotics were established. In all cases, the MIC for INH hadbeen reduced from 512 to 8 μg/ml, a value lower than that of thesensitive strain MC² 155 (32 μg/ml). This hypersensitive phenotypesuggested that the recombinant clones might be overproducing an enzymecapable of enhancing INH-toxicity. Enzymological studies showed thatthese transformants all produced about 2-fold more peroxidase andcatalase than the wild type strain M2155, which is INH-sensitive.

In addition to INH, many MDR-strains of M. tuberculosis are no longersensitive to rifampicin, streptomycin, ethambutol and pyrazinamide. Toexamine the possibility that there might be a relationship betweenresistance to INH and these compounds, the MICs of several drugs forvarious M. smegmatis strains and their pBH4 transformants weredetermined, but no differences were found.

Cloning the M. tuberculosis catalase gene

A 45-mer oligonucleotide probe was designed based on the primarysequences of highly conserved regions in the catalase-peroxidaseenzymes, HPI, of E. coli (Triggs-Raine et al., 1989), and Bacillusstearothermophilus (Loprasert et al., 1988). When genomic blots of M.tuberculosis DNA were probed with this oligonucleotide, specific bandswere detected in most cases. As KpnI generated a unique fragment of 4.5kb that hybridized strongly, this enzyme was used to produce a sizeselected library in pUC19.

Upon screening with the oligonucleotide probe, an appropriate clone,pYZ55, was obtained. A restriction map of the insert DNA is presented inFIG. 1 where it can be seen that this corresponds exactly to part ofpBH4. Independent confirmation was also obtained by cross-hybridization.

By means of various subcloning experiments the smallest fragmentexpressing M. tuberculosis catalase-peroxidase activity in E. coli wasfound to be a 2.5 kb EcoRV-KpnI fragment which, as expected, contained acleavage site for BamHI. Partial DNA sequence analysis showed that thekatG gene carried by pYZ56 encodes a catalase-peroxidase enzyme that ishighly homologous to the HPI enzymes of E. coli and M.stearothermophilus: ##STR1## (FIG. 2; Triggs-Raine et al., 1988);(Loprasert et al., 1988). Identical residues are indicated by *. HPIactivity was detected in both E. coli and M. smegmatis by staining (seebelow).

Catalase-peroxidase involvement in INH-sensitivity

Having cloned the M. tuberculosis katG gene, it was of immediateinterest to investigate the genetic basis of the association betweencatalase-negativity and isoniazid resistance. A series of constructs wasestablished in the shuttle vector pBAK14 and used to transform theINH-resistant M. smegmatis mutant BH1. Only those plasmids carrying acomplete katG gene (SEQ ID NO:45) produced HPI and restoredINH-sensitivity. The smallest of these, pBAK14, carried a 2.5 kbEcoRV-KpnI fragment thus demonstrating that the 2 kb region upstream ofkatG was not involved, and that catalase-peroxidase activity alone wassufficient to render mycobacteria susceptible to INH.

Cell-free extracts were separated by non-denaturating polyacrylamide gelelectrophoresis and stained for peroxidase and catalase activity. Underthese conditions, the M. tuberculosis enzyme gave two bands ofperoxidase activity (lane 1) which comigrated with catalase activity(Heym et al., 1992).

When introduced into E. coli, the katG gene (SEQ ID NO:45) directed thesynthesis of the same proteins, whereas pYZ56 produced proteins slightlylarger in size. This is due to the construction of an in-framelacZ::katG gene fusion. Activity stains were also performed with cellextracts of M. smegmatis. The presence of the katG gene (SEQ ID NO:45)from the M. tuberculosis in BH1 led to the production ofcatalase-peroxidase enzyme, which displayed the same electrophoreticmobility as the enzyme made in M. tuberculosis, or in E. coli, and thenative HPI of M. smegmatis.

Basis of INH-resistance in M. tuberculosis

It has been known for many years that a subset of INH-resistant strains,particularly those resistant to the highest drug concentrations, are oflower virulence in the guinea pig and devoid of catalase activity.Genomic DNA was prepared from several clinical isolates of M.tuberculosis and analyzed by Southern blotting using the 4.5 kb KpnIfragment (SEQ ID NO:45) as a probe. In two highly resistant strains,B1453 and 24, the catalase gene has been deleted from the chromosomewhereas in others (FIG. 3), such as strain 12, showing low levelresistance it is still present but not expressed. Additional studiesshowed that the region immediately prior to katG (SEQ ID NO:45) washighly prone to rearrangements.

M. tuberculosis HPI renders E. coli sensitive to INH

To determine whether the HPI enzyme of M. tuberculosis could confer INHsensitivity on E. coli, a series of catalase mutants was transformedwith pYZ56 and the MICs determined. Wild type strains were notsusceptible to INH, but mutants lacking both endogenous catalaseactivities, but harboring pYZ56, showed growth inhibition when highlevels of INH (500 μg/ml) were present, whereas untransformed strainswere insensitive.

For purposes of this invention, a plasmid containing the restrictionendonuclease map shown in FIG. 1 was deposited in strain with theNational Collection of Cultures of Microorganisms (C.N.C.M.) of theInstitut Pasteur, in Paris, France on May 18, 1992, under culturecollection accession No. I-1209. This plasmid contains the nucleic acidsequence of the invention, namely, the 4.5 kb KpnI--KpnI fragment (SEQID NO:45) of plasmid pYZ56 having the BamHI cleavage site in thefragment.

In general, the invention features a method of detecting the presence ofisoniazid-resistant Mycobacterium tuberculosis in a sample includingproviding at least one DNA or RNA probe capable of selectivelyhybridizing to isoniazid-sensitive Mycobacterium tuberculosis DNA toform detectable complexes. Detection is carried out with a sample underconditions which allow the probe to hybridize to isoniazid-sensitiveMycobacterium tuberculosis DNA present in the sample to form hybridcomplexes and detecting the hybrid complexes as an indication of thepresence of isoniazid-sensitive Mycobacterium tuberculosis in thesample. (The term "selectively hybridizing"), as used herein, refers toa DNA or RNA probe which hybridizes only to isoniazid-sensitiveMycobacterium tuberculosis and not to isoniazid-insensitiveMycobacterium tuberculosis.) The sample can be comprised of theMycobacterium tuberculosis cells or a portion of the cells or cellcontents enriched in Mycobacterium tuberculosis nucleic acids,especially DNA. Hybridization can be carried out using conventionalhybridization reagents. The particular hybridization conditions have notbeen found to be critical to the invention.

More particularly, DNA sequences from Mycobacterium tuberculosis can beanalyzed by Southern blotting and hybridization. The techniques used forthe present invention are described in Maniatis et al. (1989). DNAfragments can be separated on agarose gels and denatured in situ. Thefragments can then be transferred from the gel to a water insolublesolid, porous support, such as a nitrocellulose filter, a nylonmembrane, or an activated cellulose paper, where they are immobilizedfor example, the Hybond® membrane commercialized by Amersham can beused. After prehybridization to reduce non-specific hybridization withthe probe, the solid support is hybridized to the nucleic acid probe ofthe invention. The solid support is washed to remove unbound and weaklybinding probe, and the resulting hybrid duplex molecule is examined. Aconvenient alternative approach is to hybridize oligonucleotides to theDNA denatured in the gel.

The amount of labeled probe which is present in the hybridizationsolution will vary widely, depending upon the nature of the label, theamount of the labeled probe which can reasonably bind to the filter, andthe stringency of the hybridization. Generally, substantial excesses ofthe probe over stoichiometric will be employed to enhance the rate ofbinding of the probe to the fixed DNA.

Various degrees of stringency of hybridization can be employed. The moresevere the conditions, the greater the complementary that is requiredfor hybridization between the probe and the polynucleotide for duplexformation. Severity can be controlled by temperature, probeconcentration, probe length, ionic strength, time, and the like.Conveniently, the stringency of hybridization is varied by changing thepolarity of the reactant solution. Temperatures to be employed can beempirically determined or determined from well known formulas developedfor this purpose.

Unlike Southern hybridization where DNA fragments are transferred froman agarose gel to a solid support, the method of the invention can alsobe carried out by oligonucleotide hybridization in dried agarose gels.In this procedure, the agarose gel is dried and hybridization is carriedout in situ using an oligonucleotide probe of the invention. Thisprocedure is preferred where speed of detection and sensitivity may bedesirable. The procedure can be carried out on agarose gels containinggenomic or cloned DNA of Mycobacterium tuberculosis.

In addition, the method of this invention can be carried out by transferof Mycobacterium tuberculosis DNA from polyacrylamide gels to nylonfilters by electroblotting. Electroblotting may be desirable where timeis of the essence, because electroblotting is typically faster thancapillary blotting developed to transfer DNA from agarose gels. Thismethod can be carried out in conjunction with UV-crosslinking. Thepolyacrylamide gel containing the samples to be tested is placed incontact with an appropriately prepared nylon filter. These are thensandwiched into an electroblotting apparatus and the DNA is transferredfrom the gel onto the filter using electric current. After a bufferrinse, the filter is ready to be prehybridized and hybridized orUV-crosslinked.

The method of the invention can be carried out using the nucleic acidprobe of the invention for detecting Mycobacterium tuberculosisresistant to isoniazid. The probe can be detected using conventionaltechniques.

The method of the invention can also detect point mutations in the KatGgene (SEQ ID NO:45), as well as a partial deletion of that gene.

The nucleotides of the invention can be used as probes for the detectionof a nucleotide sequence in a biological sample of M. tuberculosis. Thepolynucleotide probe can be labeled with an atom or inorganic radical,most commonly using a radionuclide, but also perhaps with a heavy metal.labels include ³² P, ³ H, ¹⁴ C, or the like. Any radioactive label canbe employed, which provides for an adequate signal and has sufficienthalf-life. Other labels include ligands that can serve as a specificbinding member to a labeled antibody, fluorescers, chemiluminescers,enzymes, antibodies which can serve as a specific binding pair memberfor a labeled ligand, and the like. The choice of the label will begoverned by the effect of the label on the rate of hybridization andbinding of the probe to the DNA or RNA. It will be necessary that thelabel provide sufficient sensitivity to detect the amount of DNA or RNAavailable for hybridization.

In preferred embodiments of the invention, the probe is labeled with aradioactive isotope, e.g., ³² P or ¹²⁵ I, which can be incorporated intothe probe, e.g., by nick-translation.

In other preferred embodiments, the probe is labeled with biotin, whichreacts with avidin to which is bonded a chemical entity which, when theavidin is bonded to the biotin, renders the hybrid DNA complex capableof being detected, e.g., a fluorophore, which renders the hybrid DNAcomplex detectable fluorometrically; an electron-dense compound capableof rendering the hybrid DNA complexes detectable by an electronmicroscope; an antibody capable of rendering the hybrid DNA complexesimmunologically detectable; or one of a catalyst/substrate pair capableof rendering the hybrid DNA complexes enzymatically detectable. Prior tocontacting the bacteria with the probe, the M. tuberculosis bacteria canbe lysed to release their DNA, which is then denatured and immobilizedon an appropriate solid, DNA-binding support, such as a nitrocellulosemembrane.

Another detection method, which does not require the labeling of theprobe, is the so-called sandwich hybridization technique. In this assay,an unlabeled probe, contained in a single-stranded vector, hybridizes toisoniazid-sensitive Mycobacterium tuberculosis DNA, and a labeled,single-stranded vector, not containing the probe, hybridizes to theprobe-containing vector, labeling the whole hybrid complex.

The sequences of the invention were derived by dideoxynucleotidesequencing. The base sequences of the nucleotides are written in the5'→3' direction. Each of the letters shown is a conventional designationfor the following nucleotides:

A Adenine

G Guanine

T Thymine

C Cytosine.

The nucleotides of the invention can be prepared by the formation of3'→5' phosphate linkages between nucleoside units using conventionalchemical synthesis techniques. For example, the well-knownphosphodiester, phosphotriester, and phosphite triester techniques, aswell as known modifications of these approaches, can be employed.Deoxyribonucleotides can be prepared with automatic synthesis machines,such as those based on the phosphoramidite approach. Oligo- andpolyribonucleotides can also be obtained with the aid of RNA ligaseusing conventional techniques.

The nucleotides of the invention are in a purified form. For instance,the nucleotides are free of human blood-derived proteins, human serumproteins, viral proteins, nucleotide sequences encoding these proteins,human tissue, and human tissue components. In addition, it is preferredthat the nucleotides are free of other nucleic acids, extraneousproteins and lipids, and adventitious microorganisms, such as bacteriaand viruses.

This invention of course includes variants of the nucleotide sequencesof the invention or serotypic variants of the probes of the inventionexhibiting the same selective hybridization properties as the probesidentical herein.

The nucleotide sequences of the present invention can be employed in aDNA amplification process known as the polymerase chain reaction (PCR) .See, e.g. Kwok et al. (1987). PCR is advantageous because this techniqueis rapid.

DNA primer pairs of known sequence positioned 10-300 base pairs apartthat are complementary to the plus and minus strands of the DNA to beamplified can be prepared by well known techniques for the synthesis ofoligonucleotides. One end of each primer can be extended and modified tocreate restriction endonuclease sites when the primer is annealed to thePBMC DNA. The PCR reaction mixture can contain the PBMC DNA, the DNAprimer pairs, four deoxyribonucleoside triphosphates, MgCl₂, DNApolymerase, and conventional buffers. The DNA can be amplified for anumber of cycles. It is generally possible to increase the sensitivityof detection by using a multiplicity of cycles, each cycle consisting ofa short period of denaturation of the PBMC DNA at an elevatedtemperature, cooling of the reaction mixture, and polymerization withthe DNA polymerase.

Amplified sequences can be detected by the use of a technique termedoligomer restriction (OR). Single-strand conformation polymorphism(SSCP) analysis can be used to detect DNA polymorphisms and pointmutations in a variety of positions in DNA fragments. See, Saiki et al.(1985); Orita et al. (1989). For example, after amplification, a portionof the PCR reaction mixture can be separated and subjected tohybridization with an end-labeled nucleotide probe, such as a ³² plabelled adenosine triphosphate end-labeled probe. In OR, an end-labeledoligonucleotide probe hybridizes in solution to a region of theamplified sequence and, in the process, reconstitutes a specificendonuclease site. Thus, hybridization of the labeled probe with theamplified katG sequence (SEQ ID NO:45) yields a double-stranded DNA formthat is sensitive to selective restriction enzyme digestion. Afterrestriction with an endonuclease, the resulting samples can be analyzedon a polyacrylamide gel, and autoradiograms of the portion of the gelwith the diagnostic labeled fragment can be obtained. The appearance ofa diagnostic fragment (e.g., 10-15 bases in length) in the autoradiogramindicates the presence of katG sequences (SEQ ID NO:45) in the PBMCs.

Since it may be possible to increase the sensitivity of detection byusing RNA instead of chromosomal DNA as the original template, thisinvention contemplates using RNA sequences that are complementary to theDNA sequences described herein. The RNA can be converted tocomplementary DNA with reverse transcriptase and then subjected to DNAamplification.

EXPERIMENTAL PROCEDURES

Bacterial strains and plasmids

Table 1 outlines the properties of the bacterial strains and plasmidsused in this invention.

                  TABLE 1                                                         ______________________________________                                        Bacterial Strains and Plasmids                                                Strains/plasmids                                                                            Characteristics                                                 ______________________________________                                        E. coli NM554                                                                 E. coli TG1   supE hsd5 thi delta (lac-proAB)  traD36                                       proAB + lacI.sup.g  lacZ delta M15!                             E. coli UM2   KatE                                                            E. coli UM255 KatE                                                            M. tuberculosis H37Rv                                                                       Virulent strain originally isolated                                           from tuberculosis patient                                       M. tuberculosis 12                                                                          Clinical isolate resistant to low                                             levels of INH (1-2 μg/ml)                                    M. tuberculosis B1453                                                                       Clinical isolate resistant to high                                            levels of INH (>50 μg/ml)                                    M. tuberculosis 24                                                                          Clinical isolate resistant to high                                            levels of INH (>50 μg/ml)                                    M. tuberculosis 79112                                                                       Clinical isolate sensitive to INH                               M. tuberculosis 12646                                                                       Clinical isolate sensitive to INH                               M. tuberculosis 79665                                                                       Clinical isolate sensitive to INH                               M. smegmatis MC.sup.2 155                                                                   MC26 het                                                        M. smegmatis BH1                                                                            MC2155 het katG                                                 Plasmids                                                                      pBH4          Shuttle cosmid, katG+, based on pYUB18                          pBH5          Deleted version of pBN4, katG+, (7 kb-                                        EcoRI)                                                          pYZ55         pUC19 derivative with 4.5 kb KpnI                                             fragment, kat+                                                  pYZ56         pUC19 derivative with 2.5 kb EcoRV-KpnI                                       fragment (kat+)                                                 pYZ57         pUC19 derivative with 3.1 kb KpnI-BamHI                                       fragment, kat-                                                  pBAK14        Mycobacterial shuttle vector (Zhang                                           et al., 1991)                                                   pBAK15        Mycobacterial shuttle vector carrying                                         4.5 kb KpnI fragment (kat+)                                     pBAK16        Mycobacterial shuttle-vector carrying                                         2.5 kb EcoRV-KpnI fragment (kat*)                               pBAK17        Mycobacterial shuttle vector carrying                                         3.1 kb KpnI-BamHI fragment (kat-)                               ______________________________________                                    

The M. tuberculosis H37 RV genomic library was constructed in theshuttle cosmid pYUB18 (Snapper et al., 1988) and kindly supplied by Dr.W. R. Jacobs. Other shuttle vectors employed were pYUB12 (Snapper etal., 1988) and pBAK14 (Zhang et al., 1991).

Microbiological techniques and enzymology

Details of antibiotics used, growth conditions, enzymology and MICdeterminations can be found in Heym et al., (1992).

Nucleic acid techniques

Standard protocols were used for subcloning, Southern blotting, DNAsequencing, oligonucleotide biosynthesis, etc. (Maniatis et al., 1989;Eiglmeier et al., 1991).

Activity staining

The preparation of cell-free extracts of E. coli and mycobacteria hasbeen described (Heym et al., 1992; Zhang et al., 1991). Native proteinsamples were separated by polyacrylamide gel electrophoresis asdescribed by Laemmli (1970) except that SDS was omitted from allbuffers, samples were not boiled and betamercaptoethanol was notincluded in the sample buffer. After electrophoresis of 50 -100 μgprotein samples on 7.5% polyacrylamide gels, catalase activity wasdetected by soaking the gel in 3 mM H₂ O₂ for 20 minutes with gentleshaking. An equal volume of 2% ferric chloride and 2% potassiumferricyanide was added and clear bands of catalase activity revealed byillumination with light. Peroxidase activity was detected as brown bandsafter soaking gels in a solution containing 0.2-0.5 mg/mldiaminobenzidine and 1.5 mM H₂ O₂ for 30-120 minutes.

To generate a highly toxic compound it seems most likely that the M.tuberculosis HPI enzyme peroxidatively activates INH (Youatt, 1969;Gayathri-Devi et al., 1975). Now that the katG gene (SEQ ID NO:45) hasbeen isolated and characterized, it should be possible to make newderivatives of INH, which can be activated in a similar manner.

Example 1 Point Mutations in the katG Gene Associated with theIsoniazid-Resistance of M. Tuberculosis

It has been shown in a recent study that the catalase-peroxidase ofMycobacterium tuberculosis, encoded by the katG gene (SEQ ID NO:45), isinvolved in mediating the toxicity of the potent anti-tuberculosis drugisoniazid or INH. Mutants resistant to clinical levels of INH showreduced catalase-peroxidase activity and, in some cases, this resultsfrom the deletion of the katG gene (SEQ ID NO:45) from the chromosome.Transformation of INH-resistant strains of Mycobacterium smegmatis andM. tuberculosis with the cloned katG gene leads to restoration ofdrug-sensitivity. Expression of katG (SEQ ID NO:45) in some strains ofEscherichia coli renders this naturally resistant organism susceptibleto high concentrations of INH.

As some INH-resistant clinical isolates of M. tuberculosis have retainedan intact katG gene (SEQ ID NO:45), the molecular basis of theirresistance was investigated. This study was facilitated by theavailability of the nucleotide sequence of a 4.7 kb KpnI fragment fromthe katG region of the chromosome as this allowed primers suitable forPCR analysis to be designed. Eleven pairs of oligonucleotide primerswere synthesized (SEQ ID NOS:5-26) (see Table 2) and used to generatePCR-products, of around 280 bp, that covered the complete katG gene (SEQID NO:45) and some of the flanking sequences. In control experiments allexperiments all eleven primer pairs generated PCR products of theexpected size, highly suitable for SSCP-analysis, so a panel of 36INH-resistant strains of M. tuberculosis, of Dutch or French origin, wasexamined. Many of these strains are multidrug resistant and wereisolated from patients who were HIV-seropositive.

                                      TABLE 2                                     __________________________________________________________________________    Sequences of primer pairs used for PCR-SSCP analysis of the katG gene of      M. tuberculosis                                                                                      5' 3' Length                                                                            GIC(%)                                                                            Tm Production                            __________________________________________________________________________    Primer Pair # 1                                                               OLIGO1: GCGGGGTTATCGCCGATG                                                                           1765                                                                             1782                                                                             18  66  61.8                                                                             288                                           (SEQ ID NO:5)                                                         OLIGO2: GCCCTCGACGGGGTATTTC                                                                          2052                                                                             2034                                                                             19  63  61.9                                             (SEQ ID NO:6)                                                         Primer Pair # 2                                                               OLIGO1: AACGGCTGTCCCGTCGTG                                                                           2008                                                                             2025                                                                             18  66  61.9                                                                             300                                           (SEQ ID NO:7)                                                         OLIGO2: GTCGTGGATGCGGTAGGTG                                                                          2307                                                                             2289                                                                             19  63  61.9                                             (SEQ ID NO:8)                                                         Primer Pair # 3                                                               OLIGO1: TCGACTTGACGCCCTGACG                                                                          2169                                                                             2187                                                                             19  63  61.9                                                                             280                                           (SEQ ID NO:9)                                                         OLIGO2: CAGGTCCGCCCATGACAG                                                                           2448                                                                             2431                                                                             18  66  61.9                                             (SEQ ID NO:10)                                                        Primer Pair # 4                                                               OLIGO1: CCACAACGCCAGCTTCGAC                                                                          2364                                                                             2382                                                                             19  53  61.9                                                                             284                                           (SEQ ID NO:11)                                                        OLIGO2: GGTTCACGTAGATCAGCCCC                                                                         2647                                                                             2628                                                                             20  50  61.9                                             (SEQ ID NO:12)                                                        Primer Pair # 5                                                               OLIGO1: GCAGATGGGGCTGATCTACG                                                                         2622                                                                             2641                                                                             20  60  51.9                                                                             288                                           (SEQ ID NO:13)                                                        OLIGO2: ACCTCGATGCCGCTGGTG                                                                           2909                                                                             2892                                                                             18  66  51.9                                             (SEQ ID NO:14)                                                        Primer Pair # 6                                                               OLIGO1: GCTGGAGCAGATGGGCTTG                                                                          2829                                                                             2847                                                                             19  63  61.9                                                                             286                                           (SEQ ID NO:15)                                                        OLIGO2: ATCCACCCGCAGCGAGAG                                                                           3114                                                                             3097                                                                             18  66  61.9                                             (SEQ ID NO:16)                                                        Primer Pair # 7                                                               OLIGO1: GCCACTGACCTCTCGCTG                                                                           3088                                                                             3105                                                                             18  66  61.9                                                                             297                                           (SEQ ID NO:17)                                                        OLIGO2: CGCCCATGCGGTCGAAAC                                                                           3384                                                                             3367                                                                             18  66  61.9                                             (SEQ ID NO:18)                                                        Primer Pair # 8                                                               OLIGO1: GCGAAGCAGATTGCCAGCC                                                                          3304                                                                             3322                                                                             19  63  61.9                                                                             285                                           (SEQ ID NO:19)                                                        OLIGO2: ACAGCCACCGAGCACGAC                                                                           3588                                                                             3571                                                                             18  66  61.9                                             (SEQ ID NO:20)                                                        Primer Pair # 9                                                               OLIGO1: CAAACTGTCCTTCGCCGACC                                                                         3549                                                                             3568                                                                             20  60  61.9                                                                             281                                           (SEQ ID NO:21)                                                        OLIGO2: CACCTACCAGCACCGTCATC                                                                         3829                                                                             3810                                                                             20  60  61.9                                             (SEQ ID NO:22)                                                        Primer Pair # 10                                                              OLIGO1: TGCTCGACAACGCGAACCTG                                                                         3770                                                                             3789                                                                             20  60  61.9                                                                             280                                           (SEQ ID NO:23)                                                        OLIGO2: TCCGAGTTGGACCCGAAGAC                                                                         4049                                                                             4030                                                                             20  60  61.9                                             (SEQ ID NO:24)                                                        Primer Pair # 11                                                              OLIGO1  TACCAGGGCAAGGATGGCAG                                                                         3973                                                                             3992                                                                             20  60  61.9                                                                             280                                           (SEQ ID NO:25)                                                        OLIGO2  GCAAACACCAGCACCCCG                                                                           4252                                                                             4235                                                                             18  66  61.9                                             (SEQ ID NO:26)                                                        __________________________________________________________________________     {#courier10                                                              

Two of them gave no PCR fragment, with any of the primers used,indicating that katG (SEQ ID NO:45) had been deleted. The remaining 34strains all yielded the expected PCR products and these were analyzed onSSCP gels so that possible point mutations could be detected. In 20cases, abnormal strand mobility was observed, compared to that of katG(SEQ ID NO:45) from drug- sensitive M. tuberculosis, suggesting thatmutational events had indeed occurred. The approximate locations of themutations, as delimited by the PCR primers, are shown in Table 3.

                                      TABLE 3                                     __________________________________________________________________________    Preliminary results of PCT-SSCP analysis of katG from M. tuberculosis         strains x denotes                                                             altered mobility; del denotes deletion                                                   1   2   3   4   5   6   7                                                 MIC 1765-                                                                             2008-                                                                             2169-                                                                             2364-                                                                             2622-                                                                             2829-                                                                             3088                                       N°                                                                        Strain                                                                            (INH)                                                                             2034                                                                              2289                                                                              2431                                                                              2628                                                                              2892                                                                              3097                                                                              3367                                       __________________________________________________________________________    1/37                                                                             9488                                                                              1                           X                                           2 9577                                                                              1                                                                       3 9112                                                                              10                                                                      4 9247                                                                              1                                                                       5 9200                                                                              1                   X                                                   6 9116                                                                              1                                                                      7/31                                                                             9106                                                                              1                   X       X                                           8 9291                                                                              1                           X                                          9/10                                                                             9412                                                                              1   X                                                                  11/12                                                                            9435                                                                              1                                                                      13 9428                                                                              1           X                                                          14 9441                                                                              1           X               X                                          15/16                                                                            9444                                                                              1           X               X                                          17/18                                                                            9445                                                                              1                                                                      19/20                                                                            9330                                                                              0,2         X                                                          21/22                                                                            9420                                                                              0,2                                                                    23 9262                                                                              0,2                                                                    24/38                                                                            9523                                                                              1                   X                                                  25 9592                                                                              10          X                                                          26 9553                                                                              10                                                                     27 9485                                                                              10                  X                                                  28 9181                                                                              1           X       X                                                  29 9363                                                                              1                           X                                          30 9465                                                                              1           X                                                          32 9178                                                                              0,2                                                                    33 9468                                                                              0,2                                                                    34 9218                                                                              0,2                                                                    33 9468                                                                              0,2                                                                    34 9218                                                                              0,2                                                                    35 9503                                                                              0,2                                                                    39 9582                                                                              1                   X                                                  41 H37Rv                                                                             --                                                                     42 Ass --                                                                     43 Mou --                                                                     44 13632                                                                             >20 del del del del del del del                                        45 13549                                                                             >5  del del del del del del del                                        46 13749                                                                             >20                                                                    47 14006                                                                             10                          X                                          48 13711                                                                             >5                                                                     49 13681                                                                             >5                  X                                                  50 14252                                                                             >5                                                                     __________________________________________________________________________

On examination of a 200 bp segment of the katG gene from fiveindependent strains (9188, 9106, 9441, 9444, 9363), a single basedifference was found. This was the same in all cases, a G to Ttransversion at position 3360, resulting in the substitution of Arg-461by Leu. Thus, in addition to inactivation of katG, INH-resistance canstem from mis-sense mutations that result in an altered catalaseperoxidase. This mutation may define a site of interaction between thedrug and the enzyme. The results of DNA sequence studies with theremaining mutants are eagerly awaited.

Another conclusion that can be drawn from this study concerns themolecular basis of the multidrug resistance associated with various M.tuberculosis strains. The same mutations are found irrespective ofwhether a given patient is seropositive or seronegative for HIV. Forexample, strain 9291, isolated from an HIV-seropositive tuberculosispatient, harbors mutations conferring resistance to INH, rifampin andstreptomycin in the katG (R461L), rpoB (S425L) and rpsL (K42R) genes,respectively. The same mutations have been found separately, or incombination, in strains from HIV-seronegative individuals. This meansthat, for the set of strains studied, there is no novel, singlemechanism conferring resistance to several drugs, but rather, multidrugresistance results from the accumulation of mutations in the genes fordistinct drug targets.

Example 2 Nucleotide Sequence and Chromosomal Location of the katG Locusof M. Tuberculosis

Bacterial strains, plasmids and growth conditions

The following bacterial strains from our laboratory collections wereused in this study: M. tuberculosis H37Rv; M. smegmatis MC 155 (Snapperet al., 1990); E. coli K-12 UM2 (katE katG; Mulvey et al., 1988). Therecombinant plasmids, pYZ55 (pUC19, katG⁺), pYZ56 (pUC19, lacZ'::katG)and the shuttle clones, pBH4 (pYUB18, katG⁺), and PBAK-KK- (pBAKl4,katG⁺) have been described recently (Zhang et al. 1992, Nature) and thekatG locus of M. tuberculosis is schematized in FIG. 5. Mycobacteriawere grown at 37° C. in Middlebrook 7H9 medium, while E. coli strainswere cultivated in L-broth, with appropriate enrichments andantibiotics.

Nucleic acid techniques

Standard techniques were employed for the preparation, labelling andhybridization of DNA (Eiglmeier et al. 1991; Zhang et al. 1992, Infect.Immun.; Zhang et al. 1992, Nature). A shotgun library of randomfragments of pYZ55 was prepared in M13mp18 as described previously(Garnier et al., 1986) and sequenced using the modified dideoxytechnique (Biggin et al. 1983). Sequences were compiled and assembledinto contigs using SAP, and analyzed with NIP, SIP and PIP (Staden 1987)running on a Vax 3100 workstation. Gap closure was obtained by usingsynthetic oligonucleotide primers, synthesized on an ABI 381 apparatus,and T7 DNA polymerase (Pharmacia) to obtain sequences directly frompYZ55. To search for related sequences in the GenBank database (release73.1) the FASTA (Pearson et al. 1988) and BLAST (Altschul et al. 1990)programs were used. The PROSITE (Bairoch 1992) catalog was screened todetect possible motifs present in protein sequences and alignments weredone with the PILEUP and PRETTY modules of the GCG sequence analysispackage (Devereux et al. 1984).

Western blotting and catalase-peroxidase activity staining.

Immunoblotting of polypeptides resolved by SDS-polyacrylamide gelelectrophoresis and detection with polyclonal antibodies (purchased fromDAKO) raised against M. bovis BCG, were as described (Zhang et al. 1992,Infect. Immun., Nature, Mol. Microbiol.). Procedures for detectingcatalase and peroxidase activities have been outlined recently (Heym etal. 1992; Zhang et al. 1992 Nature).

RESULTS

Nucleotide sequence of the katG locus of M. tuberculosis.

In previous studies, the complete katG gene (SEQ ID NO:45) was clonedindependently in E. coli on a shuttle cosmid, pBH4, and on a 4.5 KpnIrestriction fragment thus giving rise to pYZ55 (FIG. 5; Zhang et al.1992, Nature). The structural gene for catalase-peroxidase wassubsequently localized to a 2.5 kb EcoRV-KpnI fragment by sub-cloning.To deduce the primary structure of this important enzyme and therebygain some insight into its putative role in the conversion of INH into apotent anti-tuberculous derivative, the nucleotide sequence of thecomplete insert from pYZ55 was determined. This was achieved by themodified dideoxy-shotgun cloning procedure (Biggin et al. 1993) and gapsbetween the contigs were closed by using specific primers.

On inspection of the resultant sequence which is shown in FIG. 6A, the4.5 kb fragment (SEQ ID NO:45) was found to contain 4795 nucleotideswith an overall dG+dC content of 64.4%. When this was analyzed for thepresence of open reading frames, with high coding-probability values, asingle candidate was detected and, from its size, composition andlocation, this was identified as katG (SEQ ID NO:45). The absence of anyadditional open reading frames, on either strand of the KpnI fragment,ruled out the possibility that genes other than katG were involved inconferring INH-susceptibility.

Further analysis of the sequence showed katG (SEQ ID NO:45) to bepreceded by two copies of a 700 bp direct repeat which were 68%identical, with the longest stretch of identity comprising 58 bp (FIG.6B)(SEQ ID NOS:46-47). When the databases were screened with thissequence no significant homologies were detected. To test thepossibility that it could correspond to a new repetitive element in M.tuberculosis, a 336 bp probe, encompassing the 58 bp repeat, was used toprobe a partially-ordered cosmid library. Positive hybridization signalswere only obtained from clones that were known to carry katG. Likewise,a single restriction fragment was detected in Southern blots of M.tuberculosis DNA digested with restriction enzymes BamHI, KpnI and RsrIIthereby indicating that this repetitive sequence is not dispersed.

Chromosomal location of katG (SEQ ID NO:45).

As part of the M. tuberculosis genome project, most of the genes forwhich probes are available have been positioned on the contig map. Fromthe series of overlapping cosmids shown in FIG. 5 it can be seen thatthe markers linked to katG are LL105 and fbpB encoding an anonymousantigen and the putative fibronectin binding protein, or alpha antigen(Matsuo et al. 1988), respectively. None of the known insertionsequences IS6110 and IS1081 (Collins et al. 1991; McAdam et al. 1990;Thierry et al. 1990, J. Clin. Microbiol.; Thierry et al. 1990, NucleicAcids Pes.), map to this area of the chromosome although the regionupstream of katG (SEQ ID NO:45) is densely populated with copies of themajor polymorphic tandem repeat, MPTR (Hermans et al. 1992; Zhang andYoung 1993).

Presence of katG (SEQ ID NO:45) homologues in other mycobacteria.

INH is exquisitely potent against members of the tuberculosis complexyet shows little, if any, activity against other mycobacteria. Todetermine whether genes homologous to katG (SEQ ID NO:45) were presentin other mycobacteria Southern blots of DNA digested with RsrII werehybridized with a probe prepared from a 2.5 kb EcoRV-KpnI restrictionfragment carrying katG (SEQ ID NO:45) from M. tuberculosis. Underconditions of high stringency good signals were obtained from M. lepraeand M. avium (FIG. 7) while barely discernible hybridization wasobserved with M. gordonae and M. szulgai. It has been shown recentlythat katG homologues are also present in M. Smegmatis and M. aurum (Heymet al. 1992).

Predicted properties of catalase-peroxidase from M. tuberculosis.

The primary structure of catalase-peroxidase, deduced from thenucleotide sequence of katG (SEQ ID NO:45),is shown in FIG. 6 (SEQ IDNO:49) . The enzyme is predicted to contain 735 amino acids, and todisplay a molecular weight of 80,029 daltons. A protein of this size hasbeen observed in M. tuberculosis (SEQ ID NO:48), and both recombinant M.smegmatis and E. coli (SEQ ID NO:49)(see below).

Primary structures are available for several other bacterialcatalase-peroxidases including those from E. coli (SEQ ID NO:49),Salmonella tyohimurium (SEQ ID NO:50), and Bacillus stearothermophilus(SEQ ID NO:51)(Loewen et al. 1990; Loprasert et al. 1988; Triggs-Raineet al. 1988) and these have been shown to be distantly related to yeastcytochrome c peroxidase (SEQ ID NO:52) (Welinder 1991). As the crystalstructure of the latter has been determined (Finzel et al. 1984) thiscan be used to interpret the sequences of the bacterial enzymes. The M.tuberculosis enzyme (SEQ ID NO:48) shows 53.3% conservation with theenterobacterial HPI enzymes, and shares 45.7% identity with the proteinfrom B. stearothermophilus (SEQ ID NO:51). An alignment of the sequencesof these four enzymes is shown in FIG. 8 (SEQ ID NOS:48-51), along withthat of yeast cytochrome c peroxidase (SEQ ID NO:52)(Welinder 1991). Itis apparent that the NH₂ terminus, which has no counterpart in the yeastenzyme, is the most divergent part suggesting that this domain of theprotein can tolerate extensive deviation and is not required forcatalysis. Experimental support for this interpretation is provided inthe form of a LacZ-KatG fusion protein which contains an additional 40amino acid residues (FIG. 9, lane 6; Zhang et al. 1992, Nature).Addition of this NH₂ -terminal segment does not noticeably interferewith either the catalase or peroxidase reactions effected by KatG (SEQID NO:48) as judged by activity staining (Zhang et al. 1992, Nature).

Bacterial catalase-peroxidases are believed to have evolved by means ofa gene duplication event and consist or two modules, both showinghomology to the yeast enzyme, fused to a unique NH₂ -terminal sequenceof about 50 amino acid residues (Welinder 1991). The M. tuberculosisenzyme (SEQ ID NO:48) conforms to this pattern and when searched forinternal homology using SIP (Staden 1987) it was clear that the regionbetween residues 55-422 was related to the carboxy terminal domain,consisting of amino acids 423-735. Only one of the two active sitemotifs typical of peroxidases, present in the PROSITE catalog (Bairoch1992) was found when the M. tuberculosis catalase-peroxidase primarystructure (SEQ ID NO:48) was screened as there are two deviations fromthe consensus around His²⁶⁹ where the second motif should be. (Consensuspattern for peroxidase 1: DET!- LIVMT!-x(2)- LIVM!- LIVMSTAG!- SAG!-LIVMSTAG!-H- STA!- LIVMFY! (SEQ ID NO:27); consensus pattern forperoxidase 2: SGAT!-x(3)- LIVMA!-R- LIVMA!-x- FW!-H-x- SAC! (SEQ IDNO:28); (Bairoch 1992). In addition, a possible ATP-binding motif(G-x-x-x-x-G-K-T) was detected (Balroch 1992) but as this partiallyoverlaps the active site its presence may be purely fortuitous (FIG. 8).

By analogy with yeast cytochrome c peroxidase (SEQ ID NO:52) (Welinder1991), it was possible to predict a number of structurally andcatalytically important residues all of which are located in the NH₂-terminal repeat. His²⁶⁹ should serve as the fifth ligand of theheme-iron while Asp³⁸⁰ should be its hydrogen-bonded partner. Otherresidues predicted to be involved in active site modulation and H₂ O₂binding are Arg¹⁰⁴, Trp¹⁰⁷, His¹⁰⁸, Asn¹³⁸, Thr²⁷⁴ and His²⁷⁵ (FIG. 4).According to Welinder's predictions (Welinder 1991) , Trp³²⁰ should be akey residue and be required for forming the protein-radical site(Sivaraja et al. 1989).

Antibody response to M. tuberculosis KatG (SEQ ID NO:48). To evaluatethe possible value of KatG (SEQ ID NO:48) as an immunogen, Western blotswere probed with anti-serum raised against M. bovis BCG in rabbits. Asshown in FIG. 9, the 80 kD catalase-peroxidase is one of the prominentantigens recognized in cell-free extracts of M. tuberculosis, and M.smegmatis expressing the cloned katG gene (SEQ ID NO:45)(lanes 1, 3).Likewise, on introduction of the gene into E. coli significant levels ofcatalase-peroxidase were produced a striking increase in expression wasobtained from the lacZ'-katG gene fusion which directed the synthesis ofan 85 kD fusion protein (FIG. 9, lane 6).

The aim of the present study was to determine the nucleotide sequence ofthe katG gene (SEQ ID NO:45) and to use the information obtained to tryand understand how its product (SEQ ID NO:48) mediates theINH-susceptibility of M. tuberculosis and, possibly, to explain theapparent instability of the katG region of the genome (SEQ ID NO:45).Repetitive DNA is often a source of chromosomal rearrangements andanalysis of the DNA sequence upstream of katG (SEQ ID NO:45) revealedtwo copies of a 700 bp direct repeat (SEQ ID NOS:46-47). Since thiselement appears to be confined to this locus it is unlikely to serve asa target for an event, such as homologous recombination, which couldlead to the deletion of the gene that is observed so frequently (Zhanget al. 1992, Nature; Zhang and Young 1993). Likewise, as a 70 kb stretchof the chromosome of M. tuberculosis H37Rv, encompassing katG (SEQ IDNO:45), is devoid of copies of IS6110 and IS1081, these insertionsequences do not appear to be likely sources of instability. Rather, thepresence of a cluster of major polymorphic tandem repeats, MPTR (FIG. 5;Hermans et al. 1992) situated upstream of katG (SEQ ID NO:45), suggeststhat this might act as a recombinational hotspot. It may remove both theMPTR cluster and katG (SEQ ID NO:45) (Zhang and Young 1993). Theavailability of the sequence of the katG region (SEQ ID NO:45) willallow primers suitable for the polymerase chain reaction to be designedand thus facilitate studies aimed at both rapid detection ofINH-resistance and understanding the molecular basis of chromosomalinstability.

Perhaps the most intriguing feature of the M. tuberculosiscatalase-peroxidase (SEQ ID NO:48) is its ability to mediateINH-susceptibility. In our current working hypothesis, the druginteracts with the enzyme and is converted by the peroxidase activityinto a toxic derivative which acts at a second, as yet unknown, site(Zhang et al. 1992, Nature). Although horse radish peroxidase can effectthis reaction (Pearson et al. 1988; Shoeb et al. 1985), and producehydroxyl and organic free radicals, very few bacteria, including othermycobacteria, are sensitive to INH. This is intriguing as they containgenes homologous to katG (SEQ ID NO:45) (FIG. 7). One explanation forthis could be provided by the fact that most bacterial contain twocatalases, one of which is a broad spectrum enzyme endowed withperoxidase activity, and that the second catalase, by preferentiallyremoving H₂ O₂, limits the ability of the catalase-peroxidase to oxidizeINH. As M. tuberculosis lacks the latter activity its KatG enzyme (SEQID NO:48) can convert INH to the lethal form without competition for theelectron acceptor.

Alternatively, there may be some unique features of the M. tuberculosisenzyme which promote toxicity or favor the interaction with the drug.Examination of the primary structures of the bacterialcatalase-peroxidases was not instructive in this respect as they allshare extensive sequence identities and contain two motifscharacteristic of the active sites of peroxidases. Furthermore, it hasbeen shown recently that expression of the E. coli katG gene (SEQ IDNO:49) can partially restore INH-susceptibility to drug-resistantmutants of M. tuberculosis suggesting that the endogenous enzyme may notpossess any drug-specific properties (Zhang et al. 1993). Sequencecomparison with the cytochrome c peroxidase (SEQ ID NO:52) from yeasthas provided important information about the structural and functionalorganization of the KatG protein (SEQ ID NO:48) and led to theidentification of the putatively-important catalytic residues (FIG. 8).

Now that the complete sequence of katG (SEQ ID NO:48) is available itwill be possible to test some of these hypotheses by site-directedmutagenesis and to overproduce the enzyme so that detailed analysis ofthe enzymatic reaction, and its products, can be performed in vitro.Likewise, it should be a relatively simple matter to isolate mutantsthat have retained enzymatic activity but are unable to bind or oxidizedINH. Of particular interest is the repetitive structure of the enzymeand the prediction that the NH₂ -terminal repeat contains the activesite for peroxidases. This raises the possibility that katG genes (SEQID NO:45) , mutated, or truncated at the 3'-end, could arise. It isconceivable that their products, lacking the normal COOH-terminus whichmay be required for subunit-subunit interactions (Welinder 1991), wouldbe unstable but still retain low enzyme activity. They would thus conferan intermediate level of INH-susceptibility, between that of katG⁺strains and mutants completely lacking the gene, as is often observed inclinical settings.

The invention may of course make use of a part of the above described2.5 kb EcoRV-KpnI fragment, said part being nonetheless sufficientlylong to provide for the selectivity of the in vitro detection of aMycobacterium tuberculosis resistant to isoniazid.

The invention also relates to a kit for detecting multidrug resistantvariants of M. tuberculosis wherein the kit comprises:

(a) a container means containing a probe for the gene encoding drugresistance; and

(b) a container means containing a control preparation of nucleic acid.

Needless to say that use can be made of any detection method alternativebringing into play the nucleodic sequence specific of nucleic acids of aMycobacterium resistant to isoniazid, e.g. a method using anamplification technique and primers, whereby said primers may either becontained within said specific nucleotidic sequence, in order to providefor amplification fragments containing at least a part of the nucleotidesequence of the above mentioned probe, nonetheless sufficiently long toprovide for the selectivity of the in vitro detection of a Mycobacteriumtuberculosis resistant to isoniazid, and finally detecting a possiblemutation in any of the amplified sequences.

A preferred process alternative (oligotyping) for the detection ofresistance to the selected antibiotic comprises:

fragmenting the relevant gene or part thereof likely to carry themutation into a plurality of fragments, such as by digestion of saidrelevant gene by selected restriction enzymes,

hybridizing these fragments to complementary oligonucleotide probes,preferably a series of labelled probes recognizing under stringentconditions, all of the parts of the relevant gene of a correspondingcontrol DNA of a strain non-resistant to the corresponding antibiotic,

and relating the absence of hybridization of at least one of saidoligonucleotide probes to any of the DNA fragments of the relevant geneof the mycobacterium under study as evidence of the presence of amutation and, possibly, of a resistance to the corresponding antibiotic,particularly as compared to the running of the test under the sameconditions with the same oligonucleotides on the relevant gene(s)obtained from a strain (strains) not resistant to said antibiotic.

Another process alternative (SSCP analysis, i.e. analysis of SingleStranded Conformation Polymorphisms) comprises:

digesting the DNA to be analyzed, particularly of the relevant gene,

amplifying the fragments obtained, e.g. by PCR,

recovering the amplified fragments, and

separating them from one another according to sizes, e.g. by causingthem to migrate, for instance on an electrophoretic gel, comparing thesizes of the different fragments with those obtained from the DNA(s) ofone or several control strains not resistant to the antibiotic, whichhad been subjected to a similar assay, and

relating the polymorphism possibly detected to the existence of amutation in the relevant gene, accordingly to a possible resistance tothe corresponding antibiotic of the strain from which the DNA-understudy had been obtained.

Needless to say that any other method, including classical sequencingtechniques, can resorted to for the achievement of the same purpose.

This method includes that known under the expression "oligotyping" forthe detection of polymorphisms, reference is advantageously made to themethod discloses by Orita et al. (reference was already made theretoherebefore) for the detection of polymorphisms based on the conformationof single strands.

The relevant gene in the case of resistance to isoniazid is of coursethe katG gene (SEQ ID NO:45) or a fragment thereof.

In the case of resistance to rifampicin, the relevant gene happens to bethe rpoB gene (SEQ ID NO:59) which codes for the βsub-unit of the RNApolymerases of said mycobacteria, or when only part of that gene isbeing used, preferably that part which includes the codons 400 to 450 ofthat rpoB gene.

Finally, in the case of resistance to steptomycin, the relevant genecontemplated is that of the rpsL gene (SEQ ID NO:63) that codes for theS12 protein of the small ribosome sub-unit or, when only part of saidfragment is being used, preferably that part which includes the codon atthe 43 position.

A preferred procedure, particularly in relation to the processalternative making use of PCR amplification is disclosed hereafter.

DNA is obtained from a biological sample (e.g. blood or sputum) afterremoval of the cellular debris and lysis of the bacterial cells with anappropriate lysis buffer. PCR amplication can be carried out byclassical methods, using a pair of primers, whose sequences arerespectively complementary to fragments of each of the strands of theDNA to be amplified.

The procedure may be run further as follows:

the amplification products (comprising e.g. from 100 to 300 nucleotides)are digested by means of suitable restriction endonuclease,

the ADN strands obtained from the amplification medium are subjected todenaturation,

the monostranded DNA strands are deposited on a neutral 5% polyacrylamidgel,

the monostranded DNA strands are caused to migrate on said gel by meansof electrophoresis,

the DNA fragments that migrated on the polyacrilamid gel are transferredonto a nylon membrane according to a usual electrophoretic blottingtechnique and hybridized to labelled probes, for instance ³² P labelledprobes, and

the migration distances of the DNA fragments subjected to analysis arecompared to those obtained from controls obtained under the sameconditions of amplification, digestion, denaturation electrophoresis andtransfer onto a nylon membrane, whereby said DNA had been obtained froman identical bacterial strain yet sensitive to the antibiotic understudy.

For the production of the PCR primers as well as of thepolygonucleotides probes used in the above disclosed "oligotyping"procedures, use is advantageously made of those complementary to therpoB gene (SEQ ID NO:59) of wild M. tuberculosis inserted in a plasmiddeposited under number I-12167 at the CNCM on Sep. 15, 1992.

The invention also relates more particularly to the nucleotidic sequenceof a fragment of rpsL gene (SEQ ID NO:63) of Mycobacterium tuberculosiscoding for the S12 protein of the small ribosome sub-unit, as well as tothe nucleotidic sequence of a mutated rpsL gene fragment deemedresponsible of the resistance to streptomycin.

By amplification of that nucleotidic sequence, the nucleotide sequenceof the full rpsL gene can be obtained.

Further illustration of the invention will be provided in the followingdescription of additional examples.

Example Concerning Rifampicin In Mice

The sensitivity to rifampicin has been determined in mice as disclosedby Grosset et al. (and Int. J. Lepr. 57:607-614). The cells of M. Lepraewere obtained from mouse paws according to classic procedures. Allresistant strains were able to grow in mice which received daily dosesof 20 mg/Kg of rifampicin, whereas sensitive strains were killed at lowrifampicin concentrations, less than 2 mg/Kg.

Relevant regions of the rpoB gene of extracted DNA was initiated uponusing two pairs of biotinylated primers, whose sequences appear in thefollowing table.

                  TABLE                                                           ______________________________________                                        Primer        Sequence                                                        ______________________________________                                        Brpo22        CAGGACGTCGAGGCGATCAC                                                          (SEQ ID NO:30)                                                  rpo23         AACGACGACGTGGCCAGCGT                                                          (SEq ID NO:31)                                                  Brpo24        CAGACGGTGTTTATGGGCGA                                                          (SEQ ID NO:32)                                                  rpo25         TCGGAGAAACCGAAACGCTC                                                          (SEQ ID NO:33)                                                  rpo32         TCCTCGTCAGCGGTCAAGTA                                                          (SEQ ID NO:34)                                                  rpo33         CTTCCCTATGATGACTG                                                             (SEQ ID NO:35)                                                  rpo34         GGTGATCTGCTCACTGG                                                             (SEQ ID NO:36)                                                  rpo35         GCCGCAGACGCTGATCA                                                             (SEQ ID NO:37)                                                  rpo36         TTGACCGCTGACGAGGA                                                             (SEQ ID NO:38)                                                  rpo37         GCCAGCGTCGATGGCCG                                                             (SEQ ID NO:39)                                                  ______________________________________                                    

Upon using conventional techniques, amplification products comprising310 and 710 bp were respectively obtained as shown in FIG. 1. Thelocalization of the sequences of the different primers used in the tableis also indicated on FIG. 10.

The DNAs obtained have been sequenced on the basis of the rpoB sequenceof isolates sensitive to rifampicin (SEQ ID NO:59). A plasmid containingthe sequence of that gene has been deposited at CNCM on Sep. 15, 1992under number I-1266. Biotinylated PCR products were concentrated fromthe PCR reaction mixtures by contacting with streptavidin coated beadsunder agitation. The biotinylated strands attached to the beads werethen recovered and sequenced. The sequences obtained were compared tothe sequence of the rpoB gene or a wild type strain (SEQ ID NO:59).Significant results were obtained as a result or sequencing of the wildgene (SEQ ID NO:59)(of a mycobacterium sensitive to rifampicin) and ofcorresponding sequences of the β-sub-unit of four mutant strainsresistant to rifampicin (FIG. 11).

Results were obtained starting from 102 strands obtained from patientsinfected with M.tuberculosis. Among this 102 strands 53 were sensitiveto rifampicin and 49 resistant to rifampicin. The mutation was localizedin the region 400-450 in 43 of the mutants and among the latter, themutation occurred in the region of ⁴²⁵ Ser into leu.

Example of detection of the resistance of mycobacteria to streptomycin

The culture of M.tuberbulosis strains and the test of their sensitivityto streptomycin have been carried out by the method of proportions on aLowenstein-Ierva medium (Laboratory Method for ClinicalMycobacteriology--Hugo David--Veronique Levy Frebault, M. F. Thorel,published by Institut Pasteur).

The nucleotide sequence of the rpsL gene (SEQ ID NO:61) of M.Leprae led,by sequence analogy, to the construction of two primers, ML51(CCCACCATTCAGCAGCTGGT)(SEQ ID NO:40) and ML52 (GTCGAGCGAACCGCGAATGA)(SEQID NO:41) surrounding regions including putative mutation sites liableof being responsible for the streptomycin resistance and suitable forthe PCR reaction. The DNA of the used M.tuberculosis used as a matrixhas enabled one to obtain a rpsL fragment of 306 pb (SEQ ID NO:63). Thenucleotide sequence of the sequenced fragments exhibited 28 differenceswith that of M.Leprae.

The rpsL genes or 43 strands of M.tuberculosis, among which 28 wereresistant, have been amplified both by PCR and the SSCP technique.

DNA was extracted from 200 μl aliquots of M.tuberculosis samples (inaverage 10⁴ to 10⁵ bacteria) covered by 100 μl of mineral oil by acongelation-decongelation technique (Woods and Cole, 1989 FEBS.Microbiol. Lett,65:305-308).

After electrophoresis of the DNA strands tested a mutation was shown in16 of the mutants. In order to establish the nature of the mutation inthe 16 strands under consideration, the corresponding rpsL genefragments were amplified by PCR using the ML51 (SEQ ID NO:40) and theML52 (SEQ ID NO:41) primers and their respective nucleotide sequenceswere determined.

The sequences obtained were compared to the sequence of the wild typerpsL gene (SEQ ID NO:65) . The single difference was found with the wildsequence ; codon 43, AAG, was mutated into AGG and, consequently, thelys-42 aminoacid was replaced by Arg.

The invention relates also to the "mutated" DNA fragments. They can inturn be used as hybridization probes for use for the detection insuitable hybridization procedures and for the detection of similarmutation in DNA extracted from a M.tuberbulosis strain suspected toinclude resistance to any one of the above illustrated antibiotics.

The invention further relates to kits for the resistance ofmycobacteriae to isoniazid, rifampicin or analogues thereof, andstreptomycin.

The invention further relates to a kit for the in vitro diagnostic ofthe resistance of a bacteria of a mycobacterium genus to isoniazid,characterized in that it comprises:

means for carrying out for a genic amplification of the DNA of the katGgene (SEQ ID NO:45) or of a fragment thereof,

means to bring into evidence one or several mutations on theamplification products so obtained,

a preparation of control DNA of a katG gene (SEQ ID NO:45) of a strainof said bacteria sensitive to isoniazid or of a fragment thereof,

optionally, a control preparation of a DNA of the katG gene (SEQ IDNO:45) of an isoniazid-resistant mycobacterium strain.

The invention further relates to a kit for the in vitro diagnostic ofthe resistance of a bacteria of a mycobacterium genus to rifampicin orits analogues, characterized in that it comprises:

means for carrying out for a genic amplification of the DNA of the rpoBgene (SEQ ID NO:59) or of the β-sub-unit of the RNA polymerase (SEQ IDNO:60) of said mycobacteria, or of a fragment thereof,

means to bring into evidence one or several mutations on theamplification products so obtained,

a preparation of control DNA of a rpoB gene coding for the β-sub-unit ofthe RNA polymerase of a strain of said bacteria sensitive to rifampicinor of a fragment thereof,

optionally, a control preparation of a DNA of the rpoB gene (SEQ IDNO:59) of an isoniazid-resistant mycobacterium strain.

Similarly, the invention pertains to a kit for the in vitro diagnosticsof the resistance of the M.tuberculosis to streptomycin, characterizedin that it includes:

means for carrying out a genic amplification of the rpsL gene (SEQ IDNO:63) coding for the S12 protein of the small ribosome sub-unit, orfragment thereof,

means which enable the bringing to evidence of one or several mutationson the amplification products obtained,

a control preparation of a DNA sequence of the rpsL gene (SEQ ID NO:65)coding for the S12 protein of the small sub-unit of the ribosome (SEQ IDNO:66) of a M.tuberculosis strain sensitive to streptomycin, and

optionally, a control preparation of a DNA sequence of a rpsL gene (SEQID NO:63) coding for the S12 protein of the small sun-unit or theribosome (SEQ ID NO:64) of a strain of M.tuberculosis resistant tostreptomycin.

REFERENCES CITED IN THE SPECIFICATION

Altschul, S., Gish, W., Miller, W., Myers, E., and Lipman, D. (1990). Abasic local alignment search tool. Proc. Natl. Acad. Sci. USA215:403-410.

Bekierkunst, A. & Bricker, A. (1967). Studies on the mode of action ofisoniazid on mycobacteria. Arch. Biochem. Biophys. 122:385-392.

Biggin, M. D., Gibson T. J., and Hong G. F. (1983). Buffer gradient gelsand ³⁵ S-label as an aid to rapid DNA sequence determination. Proc.Natl. Acad. Sci. USA 80:3963-3965.

Bairoch, A., (1992). Prosite: a dictionary of sites and patterns inproteins. Nucleic Acids Res. 20:2013-2018.

C. D. C. Outbreak of multidrug-resistant tuberculosis--Texas,California, and Pennsylvania. MMWR 1990, 39:369-372.

C. D. C. Nosocomial transmission of multidrug-resistant tuberculosisamong HIV-infected persons--Florida and New York 1988-1991. MMWR 1991(a)40:585-591.

C. D. C. Transmission of multidrug-resistant tuberculosis from anHIV-positive client in a residential substance abuse treatment facility.Michigan. MMWR 1991(b), 40:129-131.

Chaisson, R. E., Schecter, G. F., Theuer, C. P., Rutherford, G. W.,Echenberg, D. F., Hopewell, P. C. (1987). Tuberculosis in patients withthe acquired immunodeficiency syndrome. Am. Rev. Respir. Dis., 23:56-74.

Collins, D. M., and Stephens, D. M. (1991). Identification of aninsertion sequence, 1S1081, in Mycobacterium bovis. FEMS Microbiol.Lett. 83:11-16.

Daley, C. L., Small, P. M., Schecter, G. F., Schoolnik, G. K., McAdam,R. A., Jacobs, W. R., and Hopewell, P. C. (1992). An outbreak oftuberculosis with accelerated progression among persons infected withthe human immunodeficiency virus. An analysis usingrestriction-fragment-length-polymorphism. N. Engl. J. Med., 326:231-235.

Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive setof sequence analysis programs for the VAX. Nucl. Acids Res. 12:387-395.

Eiglmeier, K., Honore, N., and Cole, S. T. (1991). Towards theintegration of foreign DNA into the chromosome of Mycobacterium leprae.Research in Microbiology, 142:617-622.

Finzel, B. C., Poulos, T. L. and Kraut, J. (1984). Crystal structure ofyeast cytochrome C peroxidase at 1.7 Å resolution. J. Biol. Chem.259:13027-13036.

Garnier, T., and Cole, S. T., (1986). Characterization of abacteriocinogenic plasmid from Clostridium perfringens and moleculargenetic analysis of the bacteriocin-encoding gene. J. Bacteriol.,168:1189-1196.

Gayathri Devi, B., Shaila, M. S., Ramakrishnan, T., and Gopinathan, K.P. (1975). The purification and properties of peroxidase inMycobacterium tuberculosis H37RV and its possible role in the mechanismof action of isonicotinic acid hydrazide. Biochem. J., 149:187-197.

Hermans, P. W. M., van Soolingen, D. and van Embden, J. D. A. (1992).Characterization of a major polymorphic tandem repeat in Mycobacteriumtuberculosis and its potential use in the epidemiology of Mycobacteriumkansasii and Mycobacterium Ågordonae. J. Bacteriol. 174:4157-4165.

Heym, B. and Cole, S. T. (1992). Isolation and characterization ofisoniazid-resistant mutants of Mycobacterium smegmatis and M. aurum.Res. Microbiol., submitted.

Jackett, P. S., Aber, V. and Lowrie, D. (1978). J. Gen Microbiol.,104:37-45.

Kubica, G. P., Jones Jr., W. D., Abbott, V. D., Beam, R. E., Kilburn, J.O., and Cater Jr., J. C. (1966). Differential identification ofmycobacteria. I. Tests on catalase activity. Am. Rev. Rest. Dis.,94:400-405

Kwok et al., S., J. Virol. 61:1690-1694 (1987). Multidrug resistanceresults from the accumulation of mutations in the genes for distinctdrug targets.

Laemmli, U. K., (1970). Cleavage of structural proteins during theassembly of the head of bacteriophage-T4 Nature (London) 227:680-685.

Loewen, P. C., and Stauffer, G. V. (1990). Nucleotide sequence of katGof Salmonella typhimurium LT2 and characterization of its product,hydroperoxidase I. Mol. Gen. Genet. 224:147-151.

Loprasert, S., Negoro, S. and Okada, H. (1988). Thermostable peroxidasefrom Bacillus stearothermophilus. J. Gen. Microbiol., 134:1971-1976.

Loprasert, S., Negoro, S., and Okada, H. (1989). Cloning, nucleotidesequence, and expression in Escherichia coli of the Bacillusstearotherrmophilus peroxidase gene (perA). J. Bacteriol.,171:4871-4875.

Maniatis, T., Sambrook, J., and Fritsch, E. F. (1989). Molecularcloning. A laboratory manual. Second Edition 1989. Cold Spring HarborLaboratory Press.

Matsuo, K., Yamaguchi, R., Yamazaki, R. A., Tasaka, H. and Yamada, T.(1988). Cloning and expression of the Mycobacterium bovis BCG gene forextracellular a antigen. J. Bacteriol., 170:3847-3854.

Middlebrook, G. (1954). Isoniazid-resistance and catalase activity oftubercle bacilli. Am. Rev. Tuberc., 69:471-472.

Middlebrook, G., Cohn, M. L., and Schaefer, W. B. (1954). --Studies onisoniazid and tubercle bacilli. III. The isolation, drug-susceptibility,and catalase-testing of tubercle bacilli from isoniazid-treatedpatients. Am. Rev. Tuberc., 70:852-872.

Mitchison, D. A., Selkon, J. B. and Lloyd, S. (1963). J. Path. Bact.86:377-386.

Mulvey, M. R., Sorby PA, Triggs-Raine BL and Loewen PC. Gene 73:337-345(1988).

Orita, M., Iwahana, I., Kanazawa, H., Itayashi, K., and Sekiya, J.(1989). PNAS 86:2766-2770.

Pearson, W., and Lipman, D. (1988). Improved tools for biologicalsequence comparisons. Proc. Natl. Acad. Sic. USA. 85:2444-2448.

Quemard, A., Lacave, C., and Laneelle, G. (1991). Isoniazid inhibitionof mycolic acid synthesis by cell extracts of sensitive and resistantstrains of Mycobacterium aurum. Antimicrob. Ag. Chem., 35:1035-1039.

Saiki et al., R. K., Bio/Technology 3:1008-1012 (1985).

Shoeb, H. A., Bowman B. U. J., Ottolenghi, A. C., and Merola, A. J.(1985). Peroxidase-mediated oxidation of isoniazid. Antimicrobial Agentsand Chemotherapy, 27:399-403

Shoeb, H. A., Bowman, B. U. J., Ottolenghi, A. C., and Merola, A. S.(1985). Evidence for the generation of active oxygen by isoniazidtreatment of extracts of Mycobacterium tuberculosis H37Ra. AntimicrobialAgents and Chemotherapy, 27:404-407.

Sivaraja, M., Goodin, D. B., Smith, M., and Hoffman, B. M., (1989).Identification by ENDOR of Trp¹⁹¹ as the free-radical site in cytochromec peroxidase Compound Es. Science, 245:738-740.

Snapper, S. B., Lugosi, L., Jekkel, A., Melton, R. E., Kieser, T.,Bloom, B. R., and Jacobs, W. R. (1988). Lysogeny and transformation inmycobacteria: stable expression of foreign genes. Proc. Natl. Acad. Sci.USA, 85:6987-6991.

Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T., and Jacobs, W.R. (1990). Isolation and characterization of efficient plasmidtransformation mutants of Mycobacterium smegmatis. Mol. Microbiol.,4:1911-1919.

Snider, D. (1989). Rev. Inf. Dis., S335.

Snider Jr., D. E. and Roper, W. L. (1992). The new tuberculosis. The NewEngland Journal of Medicine, 326:703-705.

Sriprakash, K. S. and Ramakrishnan, T. (1970). Isoniazid-resistantmutants of Mycobacterium tuberculosis H37Rv: Uptake of isoniazid and theproperties of NADase inhibitor. J. Gen. Microbiol., 60:125-132.

Staden, P. (1987). Computer handling of sequence projects. In Nucleicacid and protein sequence analysis: A practical approach. Bishop, M. J.and Rawlings, C. J. (eds.) Oxford: IRL Press, pp. 173-217.

Thierry, D., Brisson-Noel, A., Vincent-Levy-Frebault, V., Nguyen, S.,Guesdon, J., and gicquel, B. (1990). Characterization of a Mycobacteriumtuberculosis insertion sequence, IS6110, and its application indiagnosis. S. Clin. Microbiol., 28:2668-2673.

Thierry, D., Cave, M. D., Sisenach, K. D., Crawford, S. T., Bates, S.H., Gicquel, B., and Guesdon, J. L. (1990). IS6110, an IS-like elementof Mycobacterium tuberculosis complex. Nucleic Acids Res., 18:188.

Triggs-Raine, B. L., Doble, B. W., Mulvey, M. R., Sorby, P. A., andLoewen, P. C. (1988). Nucleotide sequence of katG, encoding catalase HPIof Escherichia coli. J. Bacteriol., 170:4415-4419.

Wayne, L. G. and Diaz, G. A. (1986). Analyt. Biochem. 157:89-92.

Welinder, K. G. (1991). Bacterial catalase-peroxidases are geneduplicated members of the plant peroxidase superfamily. Biochim.Biophys. Acta 1080:215-220.

Winder, F. and Collins, P. (1968). The effect of isoniazid onnicotinamide nucleotide levels in Mycobacterium bovis, strain BCG. Amer.Rev. Pespir. Dis., 97:719-720.

Winder, F. and Collins, P. (1969). The effect of isoniazid onnicotinamide nucleotide concentrations in tubercle bacilli. Amer. Rev.Respir. Dis., 100:101-103.

Winder, F. and Collins, P. (1968). Inhibition by isoniazid of synthesisof mycolic acids in Mycobacterium tuberculosis, J. Gen. Microbiol.,63:41-48.

Youatt, J. (1969). A review of the action of isoniazid. Am. Rev. Respir.Dis., 99:729-749.

Zhang, Y., Garbe, T., and Young, D. (1993). Transformation with katGrestores isoniazid-sensitivity in Mycobacterium tuberculosis isolatesresistant to a range of drug concentrations. Mol. Microbiol., submitted.

Zhang, Y., and Young, D. B. (1993) Characterization of a variablegenetic element from the katG region of Mycobacterium tuberculosis--inpreparation.

Zhang, Y., Lathigra, R., Garbe, T., Catty, D., and Young, D. (1991)Genetic analysis of superoxide dismutase, the 23 kilodalton antigen ofMycobacterium tuberculosis. Mol. Microbiol., 5:381-391.

Zhang, Y., Heym, B., Allen, B., Young, D., and Cole, S. T. (1992). Thecatalase-peroxidase gene and isoniazid resistance of Mycobacteriumtuberculosis. Nature. 358:591-593.

Zhang, Y., Garcia, M. J., Lathigra, R., Allen, B., Moreno, C., vanEmbden, D. A., and Young, D. (1992). Alterations in the superoxidedismutase gene of an isoniazid-resistant strain of Mycobacteriumtuberculosis. Infect. Immun., 60:2160-2165.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 66                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TTCATCCGCATGGCCTGGCACGGCGCGGGCACCTACCGC39                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AlaProLeuAsnSerTrpProAspAsnAlaSerLeuAspLysAlaArg                              151015                                                                        ArgLeuLeuTrpProSerLysLysLysTyrGlyLysLysLeuSerTrp                              202530                                                                        AlaAspLeuIleVal                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AlaProLeuAsnSerTrpProAspAsnValSerLeuAspLysAlaArg                              151015                                                                        ArgLeuLeuTrpProIleLysGlnLysTyrGlyGlnLysIleSerTrp                              202530                                                                        AlaAspLeuPheIle                                                               35                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AlaProLeuAsnSerTrpProAspAsnAlaAsnLeuAspLysAlaArg                              151015                                                                        ArgCysLeuGlyArgSerLysArgAsnThrGlyThrLysSerLeuGly                              202530                                                                        ProIleCysSer                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCGGGGTTATCGCCGATG18                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCCCTCGACGGGGTATTTC19                                                         (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AACGGCTGTCCCGTCGTG18                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GTCGTGGATGCGGTAGGTG19                                                         (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCGACTTGACGCCCTGACG19                                                         (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CAGGTCCGCCCATGACAG18                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CCACAACGCCAGCTTCGAC19                                                         (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GGTTCACGTAGATCAGCCCC20                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GCAGATGGGGCTGATCTACG20                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ACCTCGATGCCGCTGGTG18                                                          (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GCTGGAGCAGATGGGCTTG19                                                         (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ATCCACCCGCAGCGAGAG18                                                          (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GCCACTGACCTCTCGCTG18                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CGCCCATGCGGTCGAAAC18                                                          (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GCGAAGCAGATTGCCAGCC19                                                         (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      ACAGCCACCGAGCACGAC18                                                          (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CAAACTGTCCTTCGCCGACC20                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CACCTACCAGCACCGTCATC20                                                        (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      TGCTCGACAACGCGAACCTG20                                                        (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      TCCGAGTTGGACCCGAAGAC20                                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      TACCAGGGCAAGGATGGCAG20                                                        (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GCAAACACCAGCACCCCG18                                                          (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: one-of(9, 10)                                                   (D) OTHER INFORMATION: /note= "Xaa=unknown."                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      AspGluThrLeuIleValMetThrXaaXaaLeuIleValMetLeuIle                              151015                                                                        ValMetSerThrAlaGlySerAlaGlyLeuIleValMetSerThrAla                              202530                                                                        GlyHisSerThrAlaLeuIleValMetPheTyr                                             3540                                                                          (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: one-of(5, 6, 7, 19, 23)                                         (D) OTHER INFORMATION: /note= "Xaa=unknown."                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      SerGlyAlaThrXaaXaaXaaLeuIleValMetAlaArgLeuIleVal                              151015                                                                        MetAlaXaaPheTrpHisXaaSerAlaCys                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: one-of(2, 3, 4, 5)                                              (D) OTHER INFORMATION: /note= "Xaa=unknown."                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      GlyXaaXaaXaaXaaGlyLysThr                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CAGGACGTCGAGGCGATCAC20                                                        (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      AACGACGACGTGGCCAGCGT20                                                        (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      CAGACGGTGTTTATGGGCGA20                                                        (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      TCGGAGAAACCGAAACGCTC20                                                        (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      TCCTCGTCAGCGGTCAAGTA20                                                        (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      CTTCCCTATGATGACTG17                                                           (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      GGTGATCTGCTCACTGG17                                                           (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GCCGCAGACGCTGATCA17                                                           (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      TTGACCGCTGACGAGGA17                                                           (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      GCCAGCGTCGATGGCCG17                                                           (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      CCCACCATTCAGCAGCTGGT20                                                        (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      GTCGAGCGAACCGCGAATGA20                                                        (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 360 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      ATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCATCCG60                ACACTTCGCGATCACATCCGTGATCACAGCCCGATAACACCAACTCCTGGAAGGAATGCT120               GTGCCCGAGCAACACCCACCCATTACAGAAACCACCACCGGAGCCGCTAGCAACGGCTGT180               CCCGTCGTGGGTCATATGAAATACCCCGTCGAGGGCGGCGGAAACCAGGACTGGTGGCCC240               AACCGGCTCAATCTGAAGGTACTGCACCAAAACCCGGCCGTCGCTGACCCGATGGGTGCG300               GCGTTCGACTATGCCGCGGAGGTCGCGACCAGTCGACTTGACGCCCTGACGCGGGACATC360               (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      MetThrMetIleThrProSerLeuHisAlaCysArgSerThrLeuGlu                              11015                                                                         AspProHisProThrLeuArgAspHisIleArgAspHisSerProIle                              202530                                                                        ThrProThrProGlyArgAsnAlaMetProGluGlnHisProProIle                              354045                                                                        ThrGluThrThrThrGlyAlaAlaSerAsnGlyCysProValValGly                              505560                                                                        HisMetLysTyrProValGluGlyGlyGlyAsnGlnAspTrpTrpPro                              65707580                                                                      AsnArgLeuAsnLeuLysValLeuHisGlnAsnProAlaValAlaAsp                              859095                                                                        ProMetGlyAlaAlaPheAspTyrAlaAlaGluValAlaThrSerArg                              100105110                                                                     LeuAspAlaLeuThrArgAspIle                                                      115120                                                                        (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      MetSerThrSerAspAspIleHisAsnThrThrAlaThrGlyLysCys                              151015                                                                        ProPheHisGlnGlyGlyHisAspGlnSerAlaGlyAlaGlyThrThr                              202530                                                                        ThrArgAspTrpTrpProAsnGlnLeuArgValAspLeuLeuAsnGln                              354045                                                                        HisSerAsnArgSerAsnProLeuGlyGluAspPheAspTyrArgLys                              505560                                                                        GluPheSerLysLeuAspTyrTyrGlyLeuLysLysAspLeu                                    657075                                                                        (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4795 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      GGTACCGTGAGGCGATGGGTGGCCCGGGGCCCGGCTGTCTGGTAAGCGCGGCCGCAAAAC60                AGCTGTACTCTCGAATCCCAGTTAGTAACAATGTGCTATGGAATCTCCAATGACGAGCAC120               ACTTCACCGAACCCCATTAGCCACCGCGGGGCTGGCGCTCGTAGTGGCGCTGGGTGGCTG180               CGGGGGCGGGGGCGGTGACAGTCGAGAGACACCGCCATACGTGCCGAAAGCGACGACCGT240               CGACGCAACAACGCCGGCGCCGGCCGCCGAGCCACTGACGATCGCCAGTCCCATGTTCGC300               CGACGGCGCCCCGATCCCGGTGCAATTCAGCTGCAAGGGGGCCAACGTGGCCGCCACCGT360               TGACGTGGTCGTCGCCCGCGGCGAGCGAACTGGCACTCGTCGTCGATGACCCCGACGCGG420               TCGGCGGACTGTACGTGCACTGGATCGTGACCGGAATCGCCCCTGGCTCTGGCAGCACGG480               CGGATGGTCAGACTCCTGCTGGTGGGCACAGCGTGCCGAATTCTGGTGGTCGGCAAGGAT540               ACTTCGGTCCATGCCCGCCGGCGGGCACCGGGACACACCACTACCGGTTTACCCTCTACC600               ACCTTCCTGTCGCGCTCCAGCTGCCACCGGGAGCCACGGGAGTCCAAGCGGCACAGGCGA660               TAGCACAGGCCGCCAGCGACAGGCCCGGCTCGTCGGCACATTCGAAGGCTGACGCCGCGG720               CATCCCTGGCGAGGTGGTCGAAACCCTGGCTTCTCCAATTGCGCCTGGCGACAATGATCA780               ATATGGAATCGACAGTGGCGCACGCATTTCACCGGTTCGCACTGGCCATCTTGGGGCTGG840               CGCTCCCCGTGGCGCTAGTTGCCTACGGTGGCAACGGTGACAGTCGAAAGGCGGCGGCCG900               TGGCGCCGAAAGCAGCAGCGCTCGGTCGGAGTATGCCCGAAACGCCTACCGGCGATGTAC960               TGACAATCAGCAGTCCGGCATTCGCCGACGGTGCGCCGATCCCGGAACAGTACACCTGCA1020              AAGGAGCCAATATCGCGGCCTCCGTTGACCTGGTCGGCGCCGTTTGGCGGCGCACTCGTT1080              GTCGATGATCCGGACCACCTCGCGAACCTTACGTCCATTGGATCGTGATCGGGATCGCCC1140              CTGGTGCTGGCAGCAGCCGATGGTGAGACTCCCGGTGGCGGAATCAGCCTGCCGAACTCC1200              AGCGGTCAGCCCGCATACACCGGCCCCTGCCCGCCGGCGGGCACCGGGACACACCACTAC1260              CGGTTTACCCTCTACCACCTTCCTGCCGTGCCTCCACTCGCGGGACTGGCTGGGACACAA1320              GCGGCGCGGGTGATCGCGCAGGCCGCCACCATGCAGGCCCGGCTCATCGGAACATACGAA1380              GGCTGATCCACCCGCCATCCCACGATCCAGCGGCCCCGGGCGATCGGGTCCTAGCAGACG1440              CCTGTCACGCTAGCCAAAGTCTTGACTGATTCCAGAAAAGGGAGTCATATTGTCTAGTGT1500              GTCCTCTATACCGGACTACGCCGAACAGCTCCGGACGGCCGACCTGCGCGTGACCCGACC1560              GCGCGTCGCCGTCCTGGAAGCAGTGAATGCGCATCCACACGCCGACACGGAAACGATTTT1620              CGGTGCCGTGCGTTTTGCGCTGCCCGACGTATCCGGCAAGCCGTGTACGACGTGCTGCAT1680              GCCCTGACCGCCGCGGGCTTGGTGCGAAAGATCCAACCCTCGGGCTCCGTCGCGCGCTAC1740              GAGTCCAGGGTCGGCGACAACCACCATCACATCGTCTGCCGGTCTTGCGGGGTTATCGCC1800              GATGTCGACTGTGCTGTTGGCGAGGCACCCTGTCTGACGGCCTCGGACCATAACGGCTTC1860              CTGTTGGACGAGGCGGAGGTCATCTACTGGGGTCTATGTCCTGATTGTTCGATATCCGAC1920              ACTTCGCGATCACATCCGTGATCACAGCCCGATAACACCAACTCCTGGAAGGAATGCTGT1980              GCCCGAGCAACACCCACCCATTACAGAAACCACCACCGGAGCCGCTAGCAACGGCTGTCC2040              CGTCGTGGGTCATATGAAATACCCCGTCGAGGGCGGCGGAAACCAGGACTGGTGGCCCAA2100              CCGGCTCAATCTGAAGGTACTGCACCAAAACCCGGCCGTCGCTGACCCGATGGGTGCGGC2160              GTTCGACTATGCCGCGGAGGTCGCGACCAGTCGACTTGACGCCCTGACGCGGGACATCGA2220              GGAAGTGATGACCACCTCGCAGCCGTGGTGGCCCGCCGACTACGGCCACTACGGGCCGCT2280              GTTTATCCGGATGGCGTGGCACGCTGCCGGCACCTACCGCATCCACGACGGCCGCGGCGG2340              CGCCGGGGGCGGCATGCAGCGGTTCGCGCCGCTTAACAGCTGGCCCGACAACGCCAGCTT2400              GGACAAGGCGCGCCGGCTGCTGTGGCCGGTCAAGAAGAAGTACGGCAAGAAGCTCTCATG2460              GGCGGACCTGATTGTTTTCGCCGGCAACCGCTGCGCTCGGAATCGATGGGCTTCAAGACG2520              TTCGGGTTCGGCTTCGGGCGTCGACCAGTGGGAGACCGATGAGGTCTATTGGGGCAAGGA2580              AGCCACCTGGCTCGGCGATGACGGTTACAGCGTAAGCGATCTGGAGAACCCGCTGGCCGC2640              GGTGCAGATGGGGCTGATCTACGTGAACCCGGAGGCGCCGAACGGCAACCCGGACCCCAT2700              GGCCGCGGCGGTCGACATTCGCGAGACGTTTCGGCGCATGGCCATGAACGACGTCGAAAC2760              AGCGGCGCTGATCGTCGGCGGTCACACTTTCGGTAAGACCCATGGCGCCGGCCCGGCCGA2820              TCTGGTCGGCCCCGAACCCGAGGCTGCTCCGCTGGAGCAGATGGGCTTGGGCTGGAAGAG2880              CTCGTATGGCACCGGAACCGGTAAGGACGCGATCACCAGCGGCATCGAGGTCGTATGGAC2940              GAACACCCCGACGAAATGGGACAACAGTTTCCTCGAGATCCTGTACGGCTACGAGTGGGA3000              GCTGACGAAGAGCCCTGCTGGCGCTTGGCAATACACCGCCAAGGACGGCGCCGGTGCCGG3060              CACCATCCCGGACCCGTTCGGCGGGCCAGGGCGCTCCCCGACGATGCTGGCCACTGACCT3120              CTCGCTGCGGGTGGATCCGATCTATGAGCGGATCACGCGTCGCTGGCTGGAACACCCCGA3180              GGAATTGGCCGACGAGTTCCGCAAGGCCTGGTACAAGCTGATCCACCGAGACATGGGTCC3240              CGTTGCGAGATACCTTGGGCCGCTGGTCCCCAAGCAGACCCTGCTGTGGCAGGATCCGGT3300              CCCTGCGGTCAGCACGACCTCGTCGGCGAAGCAGATTGCCAGCCTTAAGAGCCAGATCCG3360              GGCATCGGGATTGACTGTCTCACAGCTAGTTTCGACCGCATGGGCGGCGGCGTCGTCGTT3420              CCGTGGTAGCGACAAGCGCGGCGGCGCCAACGGTGGTCGCATCCGCCTGCAGCCACAACT3480              CGGGTGGGAGGTCAACGACCCCGACGGATCTGCGCAAGGTCATTCGCACCCTGAAGAGAT3540              CCAGGAGTCATTCACTCGGCGCGGGAACATCAAAGTGTCCTTCGCCGACCTCGTCGTGCT3600              CGGTGGCTGTGCGCCACTAGAGAAAGCAGCAAAGGCGGCTGGCCACAACATCACGGTGCC3660              CTTCACCCCGGGCCCGCACGATGCGTCGCAGGAACAAACCGACGTGGAATCCTTTGCCGT3720              GCTGGAGCCCAAGGCAGATGGCTTCCGAAACTACCTCGGAAAGGGCAACCGTTGCCGGCC3780              GAGTACATCGCTGCTCGACAAGGCGAACCTGCTTACGCTCAGTGCCCCTGAGATGACGGT3840              GCTGGTAGGTGGCCTGCGCGTCCTCGGCGCAAACTACAAGCGCTTACCGCTGGGCGTGTT3900              CACCGAGGCCTCCGAGTCACTGACCAACGACTTCTTCGTGAACCTGCTCGACATGGGTAT3960              CACCTGGGAGCCCTCGCCAGCAGATGACGGGACCTACCAGGGCAAGGATGGCAGTGGCAA4020              GGTGAAGTGGACCGGCAGCCGCGTGGACCTGGTCTTCGGGTCCAACTCGGAGTTGCGGGC4080              GCTTGTCGAGGTCTATGCGCCGATGACGCGGCAGGCGAAGTTCGTGACAGGATTCGTCGC4140              TGCGTGGGACAAGGTGATGAACCTCGACAGGTTCGACGTGCGCTGATTCGGGTTGATCGG4200              CCCTGCCCGCCGATCAACCACAACCCGCCGCAGCACCCCGCGAGCTGACCGGCTCGCGGG4260              GTGCTGGTGTTTGCCCGGCGCGATTTGTCAGACCCCGCGTGCATGGTGGTCGCACGGACG4320              CACGAGACGGGGATGACGAGACGGGGATGAGGAGAAAGGGCGCCGAAATGTGCTGGATGT4380              GCGATCACCCGGAAGCCACCGCCGAGGAGTACCTCGACGAGGTGTACGGGATAATGCTCA4440              TGCATGGCTGGGCGGTACAGCACGTGGAGTGCGAGCGACGGCCATTTGCCTACACGGTTG4500              GTCTAACCCGGCGCGGCTTGCCCGAACTGGTGGTGACTGGCCTCTCGCCACGACGTGGGC4560              AGCGGTTGTTGAACATGCCGTCGAGGGCTCTGGTCGGTGACTTGCTGACTCCCGGTATGT4620              AGACCACCCTCAAAGCCGGCCCTCTTGTCGAAACGGTCCAGGCTACACATCCGGACGCGC4680              ATTTGTATTGTGCGATCGCCATCTTTGCGCACAAGGTGACGGCCTTGCAGTTGGTGTGGG4740              CCGACCGCGTGGTCGCTGGCCGTGGGCGGCGGACTTCGACGAAGGTCGCGGTACC4795                   (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 700 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      TTCGAAGGCTGACGCCGCGGCATCCCTGGCGAGGTGGTCGAAACCCTGGCTTCTCCAATT60                GCGCCTGGCGACAATGATCAATATGGAATCGACAGTGGCGCACGCATTTCACCGGTTCGC120               ACTGGCCATCTTGGGGCTGGCGCTCCCCGTGGCGCTAGTTGCCTACGGTGGCAACGGTGA180               CAGTCGAAAGGCGGCGGCCGTGGCGCCGAAAGCAGCAGCGCTCGGTCGGAGTATGCCCGA240               AACGCCTACCGGCGATGTACTGACAATCAGCAGTCCGGCATTCGCCGACGGTGCGCCGAT300               CCCGGAACAGTACACCTGCAAAGGAGCCAATATCGCGGCCTCCGTTGACCTGGTCGGCGC360               CGTTTGGCGGCGCACTCGTTGTCGATGATCCGGACCACCTCGCGAACCTTACGTCCATTG420               GATCGTGATCGGGATCGCCCCTGGTGCTGGCAGCAGCCGATGGTGAGACTCCCGGTGGCG480               GAATCAGCCTGCCGAACTCCAGCGGTCAGCCCGCATACACCGGCCCCTGCCCGCCGGCGG540               GCACCGGGACACACCACTACCGGTTTACCCTCTACCACCTTCCTGCCGTGCCTCCACTCG600               CGGGACTGGCTGGGACACAAGCGGCGCGGGTGATCGCGCAGGCCGCCACCATGCAGGCCC660               GGCTCATCGGAACATACGAAGGCTGATCCACCCGCCATCC700                                   (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 700 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      GGTACCGTGAGGCGATGGGTGGCCCGGGGCCCGGCTGTCTGGTAAGCGCGGCCGCAAAAC60                AGCTGTACTCTCGAATCCCAGTTAGTAACAATGTGCTATGGAATCTCCAATGACGAGCAC120               ACTTCACCGAACCCCATTAGCCACCGCGGGGCTGGCGCTCGTAGTGGCGCTGGGTGGCTG180               CGGGGGCGGGGGCGGTGACAGTCGAGAGACACCGCCATACGTGCCGAAAGCGACGACCGT240               CGACGCAACAACGCCGGCGCCGGCCGCCGAGCCACTGACGATCGCCAGTCCCATGTTCGC300               CGACGGCGCCCCGATCCCGGTGCAATTCAGCTGCAAGGGGGCCAACGTGGCCGCCACCGT360               TGACGTGGTCGTCGCCCGCGGCGAGCGAACTGGCACTCGTCGTCGATGACCCCGACGCGG420               TCGGCGGACTGTACGTGCACTGGATCGTGACCGGAATCGCCCCTGGCTCTGGCAGCACGG480               CGGATGGTCAGACTCCTGCTGGTGGGCACAGCGTGCCGAATTCTGGTGGTCGGCAAGGAT540               ACTTCGGTCCATGCCCGCCGGCGGGCACCGGGACACACCACTACCGGTTTACCCTCTACC600               ACCTTCCTGTCGCGATCCAGCTGCCACCGGGAGCCACGGGAGTCCAAGCGGCACAGGCGA660               TAGCACAGGCCGCCAGCGACAGGCCCGGCTCGTCGGCACA700                                   (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 735 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      MetProGluGlnHisProProIleThrGluThrThrThrGlyAlaAla                              151015                                                                        SerAsnGlyCysProValValGlyHisMetLysTyrProValGluGly                              202530                                                                        GlyGlyAsnGlnAspTrpTrpProAsnArgLeuAsnLeuLysValLeu                              354045                                                                        HisGlnAsnProAlaValAlaAspProMetGlyAlaAlaPheAspTyr                              505560                                                                        AlaAlaGluValAlaThrSerArgLeuAspAlaLeuThrArgAspIle                              65707580                                                                      GluGluValMetThrThrSerGlnProTrpTrpProAlaAspTyrGly                              859095                                                                        HisTyrGlyProLeuPheIleArgMetAlaTrpHisAlaAlaGlyThr                              100105110                                                                     TyrArgIleHisAspGlyArgGlyGlyAlaGlyGlyGlyMetGlnArg                              115120125                                                                     PheAlaProLeuAsnSerTrpProAspAsnAlaSerLeuAspLysAla                              130135140                                                                     ArgArgLeuLeuTrpProValLysLysLysTyrGlyLysLysLeuSer                              145150155160                                                                  TrpAlaAspLeuIleValPheAlaGlyAsnArgCysAlaArgAsnArg                              165170175                                                                     TrpAlaSerArgArgSerGlySerAlaSerGlyValAspGlnTrpGlu                              180185190                                                                     ThrAspGluValTyrTrpGlyLysGluAlaThrTrpLeuGlyAspAsp                              195200205                                                                     GlyTyrSerValSerAspLeuGluAsnProLeuAlaAlaValGlnMet                              210215220                                                                     GlyLeuIleTyrValAsnProGluAlaProAsnGlyAsnProAspPro                              225230235240                                                                  MetAlaAlaAlaValAspIleArgGluThrPheArgArgMetAlaMet                              245250255                                                                     AsnAspValGluThrAlaAlaLeuIleValGlyGlyHisThrPheGly                              260265270                                                                     LysThrHisGlyAlaGlyProAlaAspLeuValGlyProGluProGlu                              275280285                                                                     AlaAlaProLeuGluGlnMetGlyLeuGlyTrpLysSerSerTyrGly                              290295300                                                                     ThrGlyThrGlyLysAspAlaIleThrSerGlyIleGluValValTrp                              305310315320                                                                  ThrAsnThrProThrLysTrpAspAsnSerPheLeuGluIleLeuTyr                              325330335                                                                     GlyTyrGluTrpGluLeuThrLysSerProAlaGlyAlaTrpGlnTyr                              340345350                                                                     ThrAlaLysAspGlyAlaGlyAlaGlyThrIleProAspProPheGly                              355360365                                                                     GlyProGlyArgSerProThrMetLeuAlaThrAspLeuSerLeuArg                              370375380                                                                     ValAspProIleTyrGluArgIleThrArgArgTrpLeuGluHisPro                              385390395400                                                                  GluGluLeuAlaAspGluPheArgLysAlaTrpTyrLysLeuIleHis                              405410415                                                                     ArgAspMetGlyProValAlaArgTyrLeuGlyProLeuValProLys                              420425430                                                                     GlnThrLeuLeuTrpGlnAspProValProAlaValSerThrThrSer                              435440445                                                                     SerAlaLysGlnIleAlaSerLeuLysSerGlnIleArgAlaSerGly                              450455460                                                                     LeuThrValSerGlnLeuValSerThrAlaTrpAlaAlaAlaSerSer                              465470475480                                                                  PheArgGlySerAspLysArgGlyGlyAlaAsnGlyGlyArgIleArg                              485490495                                                                     LeuGlnProGlnValGlyTrpGluValAsnAspProAspGlySerAla                              500505510                                                                     GlnGlyHisSerHisProGluGluIleGlnGluSerPheThrArgArg                              515520525                                                                     GlyAsnIleLysValSerPheAlaAspLeuValValLeuGlyGlyCys                              530535540                                                                     AlaProLeuGluLysAlaAlaLysAlaAlaGlyHisAsnIleThrVal                              545550555560                                                                  ProPheThrProGlyProHisAspAlaSerGlnGluGlnThrAspVal                              565570575                                                                     GluSerPheAlaValLeuGluProLysAlaAspGlyPheArgAsnTyr                              580585590                                                                     LeuGlyLysGlyAsnArgCysArgProSerThrSerLeuLeuAspLys                              595600605                                                                     AlaAsnLeuLeuThrLeuSerAlaProGluMetThrValLeuValGly                              610615620                                                                     GlyLeuArgValLeuGlyAlaAsnTyrLysArgLeuProLeuGlyVal                              625630635640                                                                  PheThrGluAlaSerGluSerLeuThrAsnAspPhePheValAsnLeu                              645650655                                                                     LeuAspMetGlyIleThrTrpGluProSerProAlaAspAspGlyThr                              660665670                                                                     TyrGlnGlyLysAspGlySerGlyLysValLysTrpThrGlySerArg                              675680685                                                                     ValAspLeuValPheGlySerAsnSerGluLeuArgAlaLeuValGlu                              690695700                                                                     ValTyrAlaProMetThrArgGlnAlaLysPheValThrGlyPheVal                              705710715720                                                                  AlaAlaTrpAspLysValMetAsnLeuAspArgPheAspValArg                                 725730735                                                                     (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 726 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      MetSerThrSerAspAspIleHisAsnThrThrAlaThrGlyLysCys                              151015                                                                        ProPheHisGlnGlyGlyHisAspGlnSerAlaGlyAlaGlyThrThr                              202530                                                                        ThrArgAspTrpTrpProAsnGlnLeuArgValAspLeuLeuAsnGln                              354045                                                                        HisSerAsnArgSerAsnProLeuGlyGluAspPheAspTyrArgLys                              505560                                                                        GluPheSerLysLeuAspTyrTyrGlyLeuLysLysAspLeuLysAla                              65707580                                                                      LeuLeuThrGluSerGlnProTrpTrpProAlaAspTrpGlySerTyr                              859095                                                                        AlaGlyLeuPheIleArgMetAlaTrpHisGlyAlaGlyThrTyrArg                              100105110                                                                     SerIleAspGlyArgGlyGlyAlaGlyArgGlyGlnGlnArgPheAla                              115120125                                                                     ProLeuAsnSerTrpProAspAsnValSerLeuAspLysAlaArgArg                              130135140                                                                     LeuLeuTrpProIleLysGlnLysTyrGlyGlnLysIleSerTrpAla                              145150155160                                                                  AspLeuPheIleLeuAlaGlyAsnValAlaLeuGluAsnSerGlyPhe                              165170175                                                                     ArgThrPheGlyPheGlyAlaGlyArgGluAspValTrpGluProAsp                              180185190                                                                     LeuAspValAsnTrpGlyAspGluLysAlaTrpLeuThrHisArgHis                              195200205                                                                     ProGluAlaLeuAlaLysAlaProLeuGlyAlaThrGluMetGlyLeu                              210215220                                                                     IleTyrValAsnProGluGlyProAspHisSerGlyGluProLeuSer                              225230235240                                                                  AlaAlaAlaAlaIleArgAlaThrPheGlyAsnMetGlyMetAsnAsp                              245250255                                                                     GluGluThrValAlaLeuIleAlaGlyGlyHisThrLeuGlyLysThr                              260265270                                                                     HisGlyAlaGlyProThrSerAsnValGlyProAspProGluAlaAla                              275280285                                                                     ProIleGluGluGlnGlyLeuGlyTrpAlaSerThrTyrGlySerGly                              290295300                                                                     ValGlyAlaAspAlaIleThrSerGlyLeuGluValValTrpThrGln                              305310315320                                                                  ThrProThrGlnTrpSerAsnTyrPhePheGluAsnLeuPheLysTyr                              325330335                                                                     GluTrpValGlnThrArgSerProAlaGlyAlaIleGlnPheGluAla                              340345350                                                                     ValAspAlaProGluIleIleProAspProPheAspProSerLysLys                              355360365                                                                     ArgLysProThrMetLeuValThrAspLeuThrLeuArgPheAspPro                              370375380                                                                     GluPheGluLysIleSerArgArgPheLeuAsnAspProGlnAlaPhe                              385390395400                                                                  AsnGluAlaPheAlaArgAlaTrpPheLysLeuThrHisArgAspMet                              405410415                                                                     GlyProLysSerArgTyrIleGlyProGluValProLysGluAspLeu                              420425430                                                                     IleTrpGlnAspProLeuProGlnProIleTyrAsnProThrGluGln                              435440445                                                                     AspIleIleAspLeuLysPheAlaIleAlaAspSerGlyLeuSerVal                              450455460                                                                     SerGluLeuValSerValAlaTrpAlaSerAlaSerThrPheArgGly                              465470475480                                                                  GlyAspLysArgGlyGlyAlaAsnGlyAlaArgLeuAlaLeuMetPro                              485490495                                                                     GlnArgAspTrpAspValAsnAlaAlaAlaValArgAlaLeuProVal                              500505510                                                                     LeuGluLysIleGlnLysGluSerGlyLysAlaSerLeuAlaAspIle                              515520525                                                                     IleValLeuAlaGlyValValGlyValGluLysAlaAlaSerAlaAla                              530535540                                                                     GlyLeuSerIleHisValProPheAlaProGlyArgValAspAlaArg                              545550555560                                                                  GlnAspGlnThrAspIleGluMetPheGluLeuLeuGluProIleAla                              565570575                                                                     AspGlyPheArgAsnTyrArgAlaArgLeuAspValSerThrThrGlu                              580585590                                                                     SerLeuLeuIleAspLysAlaGlnGlnLeuThrLeuThrAlaProGlu                              595600605                                                                     MetThrAlaLeuValGlyGlyMetArgValLeuGlyGlyAsnPheAsp                              610615620                                                                     GlySerLysAsnGlyValPheThrAspArgValGlyValLeuSerAsn                              625630635640                                                                  AspPhePheValAsnLeuLeuAspMetArgTyrGluTrpLysAlaThr                              645650655                                                                     AspGluSerLysGluLeuPheGluGlyArgAspArgGluThrGlyGlu                              660665670                                                                     ValLysPheThrAlaSerArgAlaAspLeuValPheGlySerAsnSer                              675680685                                                                     ValLeuArgAlaValAlaGluValTyrAlaSerSerAspAlaHisGlu                              690695700                                                                     LysPheValLysAspPheValAlaAlaTrpValLysValMetAsnLeu                              705710715720                                                                  AspArgPheAspLeuLeu                                                            725                                                                           (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 729 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      MetSerThrThrAspAspThrHisAsnThrLeuSerThrGlyLysCys                              151015                                                                        ProPheHisGlnGlyGlyHisAspArgSerAlaGlyAlaGlyThrAla                              202530                                                                        SerArgAspTrpTrpProAsnGlnLeuArgValAspLeuLeuAsnGln                              354045                                                                        HisSerAsnArgSerAsnProLeuGlyGluAspPheAspTyrArgLys                              505560                                                                        GluPheSerLysLeuAspTyrTyrSerAlaLeuLysGlyAspLeuLys                              65707580                                                                      AlaLeuLeuThrAspSerGlnProTrpTrpProAlaAspTrpGlySer                              859095                                                                        TyrValGlyLeuPheIleArgMetAlaTrpHisGlyAlaGlyThrTyr                              100105110                                                                     ArgSerIleAspGlyArgGlyGlyAlaGlyArgGlyGlnGlnArgPhe                              115120125                                                                     AlaProLeuAsnSerTrpProAspThrValSerLeuAspLysAlaArg                              130135140                                                                     ArgLeuLeuTrpProIleLysGlnLysTyrGlyGlnLysIleSerTrp                              145150155160                                                                  AlaAspLeuPheIleLeuAlaGlyAsnValAlaLeuGluAsnSerGly                              165170175                                                                     PheArgThrPheGlyPheGlyAlaGlyArgGluAspValTrpGluPro                              180185190                                                                     AspLeuAspValAsnTrpGlyAspGluLysAlaTrpLeuThrHisArg                              195200205                                                                     HisProGluAlaLeuAlaLysAlaProLeuGlyAlaThrGluMetAsp                              210215220                                                                     LeuIleTyrValThrProGluGlyProAsnHisSerGlyGluProLeu                              225230235240                                                                  SerAlaAlaAlaAlaIleArgAlaThrPheGlyAsnMetGlyMetAsn                              245250255                                                                     AspGluGluThrValAlaLeuIleAlaGlyGlyHisThrLeuGlyLys                              260265270                                                                     ThrHisGlyProAlaAlaAlaSerHisValGlyAlaAspProGluAla                              275280285                                                                     AlaProIleGluAlaGlnGlyLeuGlyTrpAlaSerSerTyrGlySer                              290295300                                                                     GlyValGlyAlaAspAlaIleThrSerGlyLeuGluValValTrpThr                              305310315320                                                                  GlnThrProThrGlnTrpSerAsnTyrPhePheGluAsnLeuPheLys                              325330335                                                                     TyrGluTrpValGlnThrArgSerProAlaGlyAlaIleGlnPheGlu                              340345350                                                                     AlaValAspAlaProAspIleIleProAspProPheAspProSerLys                              355360365                                                                     LysArgXaaXaaLysProThrMetLeuValThrAspLeuThrLeuArg                              370375380                                                                     PheAspProGluPheGluLysIleSerArgArgPheLeuAsnAspPro                              385390395400                                                                  GlnAlaPheAsnGluAlaPheAlaArgAlaTrpPheLysLeuThrHis                              405410415                                                                     ArgAspMetGlyProLysAlaArgTyrIleGlyProGluValProLys                              420425430                                                                     GluAspLeuIleTrpGlnAspProLeuProGlnProLeuTyrGlnPro                              435440445                                                                     ThrGlnGluAspIleIleAsnLeuLysAlaAlaIleAlaAlaSerGly                              450455460                                                                     LeuSerIleSerGluMetValSerValAlaTrpAlaSerAlaSerThr                              465470475480                                                                  PheArgGlyGlyAspLysArgGlyGlyAlaAsnGlyAlaArgLeuAla                              485490495                                                                     LeuAlaProGlnArgAspTrpAspValAsnAlaValAlaAlaArgVal                              500505510                                                                     LeuProValLeuGluGluIleGlnLysThrThrAsnLysAlaSerLeu                              515520525                                                                     AlaAspIleIleValLeuAlaGlyValValGlyIleGluGlnAlaAla                              530535540                                                                     AlaAlaAlaArgValSerIleHisValProPheProProGlyArgVal                              545550555560                                                                  AspAlaArgHisAspGlnThrAspIleGluMetPheSerLeuLeuGlu                              565570575                                                                     ProIleAlaAspGlyPheArgAsnTyrArgAlaArgLeuAspValSer                              580585590                                                                     ThrThrGluSerLeuLeuIleAspLysAlaGlnGlnLeuThrLeuThr                              595600605                                                                     AlaProGluMetThrValLeuValGlyGlyMetArgValLeuGlyThr                              610615620                                                                     AsnPheAspGlySerGlnAsnGlyValPheThrAspLysProGlyVal                              625630635640                                                                  LeuSerThrAspPhePheAlaAsnLeuLeuAspMetArgTyrGluTrp                              645650655                                                                     LysProThrAspAspAlaAsnGluLeuPheGluGlyArgAspArgLeu                              660665670                                                                     ThrGlyGluValLysTyrThrAlaThrArgAlaAspLeuValPheGly                              675680685                                                                     SerAsnSerValLeuArgAlaLeuAlaGluValTyrAlaCysSerAsp                              690695700                                                                     AlaHisGluLysPheValLysAspPheValAlaAlaTrpValLysVal                              705710715720                                                                  MetAsnLeuAspArgPheAspLeuGln                                                   725                                                                           (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 731 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      MetGluAsnGlnAsnArgGlnAsnAlaAlaGlnCysProPheHisGlu                              151015                                                                        SerValThrAsnGlnSerSerAsnArgThrThrAsnLysAspTrpTrp                              202530                                                                        ProAsnGlnLeuAsnLeuSerIleLeuHisGlnHisAspArgLysThr                              354045                                                                        AsnProHisAspGluGluPheAsnTyrAlaGluGluPheGlnLysLeu                              505560                                                                        AspTyrTrpAlaLeuLysGluAspLeuArgLysLeuMetThrGluSer                              65707580                                                                      GlnAspTrpTrpProAlaAspTyrGlyHisTyrGlyProLeuPheIle                              859095                                                                        ArgMetAlaTrpHisSerAlaGlyThrTyrArgIleGlyAspGlyArg                              100105110                                                                     GlyGlyAlaSerThrGlyThrGlnArgPheAlaProLeuAsnSerTrp                              115120125                                                                     ProAspAsnAlaAsnLeuAspLysAlaArgArgCysTyrGlyArgSer                              130135140                                                                     LysArgAsnThrGlyThrLysSerLeuGlyProIleCysSerPheTrp                              145150155160                                                                  ArgAlaMetSerLeuLeuAsnArgTrpValGluLysArgLeuAspSer                              165170175                                                                     AlaAlaGlyProLeuThrSerGlyIleArgLysLysThrPheIleGly                              180185190                                                                     AspArgLysLysSerGlySerProLeuAsnAlaIleProValIleAla                              195200205                                                                     SerSerLysThrArgSerProArgAlaAsnGlyValAsnLeuArgGln                              210215220                                                                     ProArgArgAlaGlyArgGlnAlaGlySerLysSerArgGlyIleSer                              225230235240                                                                  AlaGluThrPheArgArgMetGlyMetAsnAspGluGluThrValAla                              245250255                                                                     LeuIleAlaGlyGlyHisThrPheGlyLysAlaHisArgGlyGlyPro                              260265270                                                                     AlaThrHisValGlyProGluProGluAlaAlaProIleGluAlaGln                              275280285                                                                     GlyLeuGlyTrpIleSerSerTyrGlyLysGlyLysGlySerAspThr                              290295300                                                                     IleThrSerGlyIleGluGlyAlaTrpThrProThrProThrGlnTrp                              305310315320                                                                  AspThrSerTyrPheAspMetLeuPheGlyTyrAspTrpTrpLeuThr                              325330335                                                                     LysSerProAlaGlyAlaTrpGlnTrpMetAlaValAspProAspGlu                              340345350                                                                     LysAspLeuAlaProAspAlaGluAspProSerLysLysValProThr                              355360365                                                                     MetMetMetThrThrAspLeuAlaLeuArgPheAspProGluTyrGlu                              370375380                                                                     LysIleAlaArgArgPheHisGlnAsnProGluGluPheAlaGluAla                              385390395400                                                                  PheAlaArgAlaTrpPheLysLeuThrHisArgAspMetGlyProLys                              405410415                                                                     ThrArgTyrLeuGlyProGluValProLysGluAspPheIleTrpGln                              420425430                                                                     AspProIleProGluValAspTyrGluLeuThrGluAlaGluIleGlu                              435440445                                                                     GluIleLysAlaLysIleLeuAsnSerGlyLeuThrValSerGluLeu                              450455460                                                                     ValLysThrAlaTrpAlaSerAlaAlaArgSerAlaThrArgIleSer                              465470475480                                                                  AlaAlaThrAsnGlyArgArgIleArgLeuAlaProGlnLysAspTrp                              485490495                                                                     GluValAsnGluProGluArgLeuAlaLysValLeuSerValLeuArg                              500505510                                                                     GlyHisProAlaArgThrAlaGluLysSerLysHisArgArgLeuAsp                              515520525                                                                     ArgLeuGlyGlyThrLeuArgTrpLysArgGlnProAlaThrProAla                              530535540                                                                     LeuMetSerLysCysHisPheSerLeuAlaAlaAlaMetArgHisLys                              545550555560                                                                  SerLysProMetSerLysAlaLeuProCysTrpAsnArgSerGlnMet                              565570575                                                                     AlaSerAlaThrIleLysSerLysSerThrArgPheArgArgLysSer                              580585590                                                                     CysSerSerThrLysProSerSerSerAlaAspArgProArgAsnAsp                              595600605                                                                     GlyLeuSerTrpArgPheAlaArgValGlyProAsnTyrArgHisLeu                              610615620                                                                     ProHisGlyValPheThrAspArgIleGlyValLeuThrAsnAspPhe                              625630635640                                                                  PheValAsnLeuLeuAspMetAsnTyrGluTrpValProThrAspSer                              645650655                                                                     GlyIleTyrGluIleArgAspArgLysThrGlyGluValArgTrpThr                              660665670                                                                     AlaThrArgValAspLeuIlePheGlySerAsnSerIleLeuArgSer                              675680685                                                                     TyrAlaGluPheTyrAlaGlnAspAspAsnGlnGluLysPheValArg                              690695700                                                                     AspPheIleAsnAlaTrpValLysValMetAsnAlaAspArgPheAsp                              705710715720                                                                  LeuValLysLysAlaArgGluSerValThrAla                                             725730                                                                        (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 293 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      ThrThrProLeuValHisValAlaSerValGluLysGlyArgSerTyr                              151015                                                                        GluAspPheGlnLysValTyrAsnAlaIleAlaLeuLysLeuArgGlu                              202530                                                                        AspAspGluTyrAspAsnTyrIleGlyTyrGlyProValLeuValArg                              354045                                                                        LeuAlaTrpHisIleSerGlyThrTrpAspLysHisAspAsnThrGly                              505560                                                                        GlySerTyrGlyGlyThrTyrArgPheLysLysGluPheAsnAspPro                              65707580                                                                      SerAsnAlaGlyLeuGlnAsnGlyPheLysPheLeuGluProIleHis                              859095                                                                        LysGluPheProTrpIleSerSerGlyAspLeuPheSerLeuGlyGly                              100105110                                                                     ValThrAlaValGluMetGlnGlyProLysIleProTrpArgCysGly                              115120125                                                                     ArgValAspThrProGluAspThrThrProAspAsnGlyArgLeuPro                              130135140                                                                     AspAlaAspLysAspAlaGlyTyrValArgThrPhePheGlnArgLeu                              145150155160                                                                  AsnMetAsnAspArgGluValValAlaLeuMetGlyAlaHisAlaLeu                              165170175                                                                     GlyLysThrHisLeuLysAsnSerGlyTyrGluGlyProTrpGlyAla                              180185190                                                                     AlaAsnAsnValPheThrAsnGluPheTyrLeuAsnLeuLeuAsnGlu                              195200205                                                                     AspTrpLysLeuGluLysAsnAspAlaAsnAsnGluGlnTrpAspSer                              210215220                                                                     LysSerGlyTyrMetMetLeuProThrAspTyrSerLeuIleGlnAsp                              225230235240                                                                  ProLysTyrLeuSerIleValLysGluTyrAlaAsnAspGlnAspLys                              245250255                                                                     PhePheLysAspPheSerLysAlaPheGluLysLeuLeuGluAsnGly                              260265270                                                                     IleThrPheProLysAspAlaProSerProPheIlePheLysThrLeu                              275280285                                                                     GluGluGlnGlyLeu                                                               290                                                                           (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 652 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      MetSerThrAspAspThrHisAsnThrThrLysCysProPheHisGln                              151015                                                                        GlyGlyHisAspGlnSerAlaGlyAlaGlyThrThrAsnArgAspTrp                              202530                                                                        TrpProAsnGlnLeuAspLeuLeuHisGlnHisSerAsnArgSerAsn                              354045                                                                        ProLeuGlyGluAspPheAspTyrLysGluPheSerLysLeuAspTyr                              505560                                                                        TyrAlaLeuLysAspLeuLysAlaLeuLeuThrGluSerGlnProTrp                              65707580                                                                      TrpProAlaAspTyrGlyTyrGlyProLeuPheIleArgMetAlaTrp                              859095                                                                        HisGlyAlaGlyThrTyrArgAspGlyArgGlyGlyAlaGlyGlyGln                              100105110                                                                     ArgPheAlaProLeuAsnSerTrpProAspAsnAlaSerLeuAspLys                              115120125                                                                     AlaArgArgLeuLeuTrpProIleLysLysTyrGlyGlnLysIleSer                              130135140                                                                     TrpAlaAspLeuPheIleLeuAlaGlyAsnValAlaLeuGluAsnPhe                              145150155160                                                                  ArgGlyPheAlaGlyArgThrGluAspValTrpGluProAspLeuAsp                              165170175                                                                     ValAsnTrpGlyGluLysAlaTrpLeuThrHisArgHisProGluLeu                              180185190                                                                     AlaLysAlaProLeuGlyAlaThrGluMetGlyLeuIleTyrValAsn                              195200205                                                                     ProGluGlyProAsnHisSerProLeuSerAlaAlaAlaAlaIleArg                              210215220                                                                     ThrPheArgMetGlyMetAsnAspGluGluThrValAlaLeuIleAla                              225230235240                                                                  GlyGlyHisThrLeuGlyLysThrHisGlyAlaGlyProAlaSerHis                              245250255                                                                     ValGlyProProGluAlaAlaProIleGluAlaGlnGlyLeuGlyTrp                              260265270                                                                     AlaSerSerTyrGlySerGlyValGlyAlaAspAlaIleThrSerGly                              275280285                                                                     GluValValTrpThrGlnThrProThrGlnTrpAsnPhePheGluAsn                              290295300                                                                     LeuPheTyrGluTrpValLeuThrLysSerProAlaGlyAlaGlnGlu                              305310315320                                                                  AlaValAspGlyAlaProAspIleIleProAspProPheAspProSer                              325330335                                                                     LysLysArgLysProThrMetLeuValThrAspLeuLeuArgPheAsp                              340345350                                                                     ProGluTyrGluLysIleSerArgArgPheLeuAsnAspProGluPhe                              355360365                                                                     GluAlaPheAlaArgAlaTrpPheLysLeuThrHisArgAspMetGly                              370375380                                                                     ProLysArgTyrIleGlyProGluValProLysGluAspLeuIleTrp                              385390395400                                                                  GlnAspProProGlnTyrProThrGluAspIleIleLeuLysAlaAla                              405410415                                                                     IleAlaAlaSerGlyLeuValSerGluLeuValSerAlaTrpAlaSer                              420425430                                                                     AlaSerThrPheArgGlyGlyAspLysArgGlyGlyAlaAsnGlyAla                              435440445                                                                     ArgLeuAlaProGlnArgAspTrpValAsnProAlaAlaArgValLeu                              450455460                                                                     ValLeuGluGluIleGlnThrLysAlaSerLeuAlaAspIleValLeu                              465470475480                                                                  GlyValValGlyGluLysAlaAlaAlaAlaAlaGlyLeuSerIleHis                              485490495                                                                     ValProPheAlaProGlyArgAspAlaArgGlnAspGlnThrAspIle                              500505510                                                                     GluMetPheLeuLeuGluProIleAlaAspGlyPheArgAsnTyrArg                              515520525                                                                     AlaLeuAspValSerThrThrGluSerLeuIleAspLysAlaGlnGln                              530535540                                                                     LeuThrLeuAlaProGluMetThrValLeuValGlyGlyMetArgVal                              545550555560                                                                  LeuGlyAsnAspGlyProAsnGlyValPheThrAspArgGlyValLeu                              565570575                                                                     AsnAspPhePheValAsnLeuLeuAspMetArgTyrGluTrpLysPro                              580585590                                                                     ThrAspLeuGluGlyArgAspArgThrGlyGluValLysTrpThrAla                              595600605                                                                     ArgAspLeuValPheGlySerAsnSerValLeuArgAlaLeuAlaGlu                              610615620                                                                     ValTyrAlaSerAspAlaGluLysPheValLysAspPheValAlaAla                              625630635640                                                                  TrpValLysValMetAsnLeuAspArgPheAspLeu                                          645650                                                                        (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      CAGTTCATGGATCAGAACAACCCTCTGTCGGGCCTGACCCACAAGCGCCGGCTGTCG57                   (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      PhePheGlySerSerGlnLeuSerGlnPheMetAspGlnAsnAsnPro                              151015                                                                        LeuSerGluIleThrHisLysArgArgIleSerAlaLeuGlyProGly                              202530                                                                        Gly                                                                           (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      PhePheGlyThrSerGlnLeuSerGlnPheMetAspGlnAsnAsnPro                              151015                                                                        LeuSerGlyLeuThrHisLysArgArgLeuSerAlaLeuGlyProGly                              202530                                                                        Gly                                                                           (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3447 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GTGCCCGGCGCGCCCAACCGAATTTCATTTGCCAAGCTCCGCGAACCGCTTGAGGTTCCG60                GGGCTACTTGATGTGCAGACTGATTCATTTGAGTGGTTGATCGGATCGCCGTGCTGGCGT120               GCAGCGGCCGCAAGCCGCGGCGATCTCAAGCCGGTGGGTGGTCTCGAAGAGGTGCTCTAC180               GAGCTGTCGCCGATCGAGGATTTCTCCGGCTCAATGTCATTGTCTTTCTCCGATCCCCGT240               TTTGACGAAGTCAAGGCGCCCGTCGAAGAGTGCAAAGACAAGGACATGACGTACGCGGCC300               CCGCTGTTCGTCACGGCCGAGTTCATCAACAACAACACCGGGGAGATCAAGAGCCAGACG360               GTGTTTATGGGCGACTTCCCTATGATGACTGAGAAGGGAACCTTCATCATCAACGGGACC420               GAGCGTGTCGTCGTTAGCCAGCTGGTGCGCTCCCCTGGAGTATACTTCGACGAGACGATC480               GACAAGTCCACAGAAAAGACGCTGCATAGTGTCAAGGTGATTCCCAGCCGCGGTGCCTGG540               TTGGAATTCGATGTCGATAAACGCGACACCGTCGGTGTCCGCATTGACCGGAAGCGCCGG600               CAACCCGTCACGGTGCTTCTCAAAGCGCTAGGTTGGACCAGTGAGCAGATCACCGAGCGT660               TTCGGTTTCTCCGAGATCATGCGCTCGACGCTGGAGAAGGACAACACAGTTGGCACCGAC720               GAGGCGCTGCTAGACATCTATCGTAAGTTGCGCCCAGGTGAGCCGCCGACTAAGGAGTCC780               GCGCAGACGCTGTTGGAGAACCTGTTCTTCAAGGAGAAACGCTACGACCTGGCCAGGGTT840               GGTCGTTACAAGGTCAACAAGAAGCTCGGGTTGCACGCCGGTGAGTTGATCACGTCGTCC900               ACGCTGACCGAAGAGGATGTCGTCGCCACCATAGAGTACCTGGTTCGTCTGCATGAGGGT960               CAGTCGACAATGACTGTCCCAGGTGGGGTAGAAGTGCCAGTGGAAACTGACGATATCGAC1020              CACTTCGGCAACCGCCGGCTGCGCACGGTCGGCGAATTGATCCAGAACCAGATCCGGGTC1080              GGTATGTCGCGGATGGAGCGGGTGGTCCGGGAGCGGATGACCACCCAGGACGTCGAGGCG1140              ATCACGCCGCAGACGCTGATCAATATCCGTCCGGTGGTCGCCGCTATCAAGGAATTCTTC1200              GGCACCAGCCAGCTGTCGCAGTTCATGGATCAGAACAACCCTCTGTCGGGCCTGACCCAC1260              AAGCGCCGGCTGTCGGCGCTGGGCCCGGGTGGTTTGTCGCGTGAGCGTGCCGGGCTAGAG1320              GTCCGTGACGTGCACCCTTCGCACTACGGCCGGATGTGCCCGATCGAGACTCCGGAGGGC1380              CCGAACATAGGTCTGATCGGTTCATTGTCGGTGTACGCGCGGGTCAACCCCTTCGGGTTC1440              ATCGAAACACCGTACCGCAAAGTGGTTGACGGTGTGGTCAGCGACGAGATCGAATACTTG1500              ACCGCTGACGAGGAAGACCGCCATGTCGTGGCGCAGGCCAACTCGCCGATCGACGAGGCC1560              GGCCGTTCCTCGAGCCGCGCGTGTTGGGTGCGCCGCAAGGCGGGCGAGGTGGAGTACGTG1620              GCCTCGTCCGAGGTGGATTACATGGATGTCTCGCCACGCCAGATGGTGTCGGTGGCCACA1680              GCGATGATTCCGTTCCTTGAGCACGACGACGCCAACCGTGCCCTGATGGGCGCTAACATG1740              CAGCGCCAAGCGGTTCCGTTGGTGCGCAGCGAACGACCGTTGGTGGGTACCGGTATGGAG1800              TTGCGCGCGGCCATCGACGCTGGCCACGTCGTCGTTGCGGAGAAGTCCGGGGTGATCGAG1860              GAGGTTTCCGCCGACTACATCACCGTGATGGCCGATGACGGCACCCGGCGGACTTATCGG1920              ATGCGTAAGTTCGCGCGCTCCAACCACGGCACCTGCGCCAACCAGTCCCCGATCGTGGAT1980              GCGGGGGATCGGGTCGAGGCCGGCCAAGTGATTGCTGACGGTCCGTGCACTGAGAACGGC2040              GAGATGGCGTTGGGCAAGAACTTGCTGGTGGCGATCAATGCCGTGGGAGGGTCAACAACT2100              AACGAGGATGCGATCATCCTGTCTAACCGACTGGTCGAAGAGGACGTGCTTACTTCGATT2160              CACATTGAGGAGCATGAGATCGACGCCCGTGACACCAAGCTGGGTGCTGAGGAGATCACC2220              CGGGACATTCCCAACGTCTCCGATGAGGTGCTAGCCGACTTGGACGAGCGGGGCATCGTG2280              CGGATTGGCGCGGAGGTTCGTGACGGTGATATCCTGGTTGGCAAGGTCACCCCGAAGGGG2340              GAAACTGAGCTGACACCGGAAGAGCGGTTGCTGCGGGCGATCTTCGGCGAAAAGGCCCGC2400              GAGGTCCGTGACACGTCGCTGAAGGTGCCACACGGCGAATCCGGCAAGGTGATCGGCATT2460              CGGGTGTTCTCCCATGAGGATGACGACGAGCTGCCCGCCGGCGTCAACGAGCTGGTCCGT2520              GTCTACGTAGCCCAGAAGCGCAAGATCTCTGACGGTGACAAGCTGGCTGGGCGGCACGGC2580              AACAAGGGCGTGATCGGCAAGATCCTGCCTGCCGAGGATATGCCGTTTCTGCCAGACGGC2640              ACCCCGGTGGACATCATCCTCAACACTCACGGGGTGCCGCGGCGGATGAACGTCGGTCAG2700              ATCTTGGAAACCCACCTTGGGTGGGTAGCCAAGTCCGGCTGGAAGATCGACGTGGCCGGC2760              GGTATACCGGATTGGGCGGTCAACTTGCCTGAGGAGTTGTTGCACGCTGCGCCCAACCAG2820              ATCGTGTCGACCCCGGTGTTCGACGGCGCCAAGGAAGAGGAACTACAGGGCCTGTTGTCC2880              TCCACGTTGCCCAACCGCGACGGCGATGTGATGGTGGGCGGCGACGGCAAGGCGGTGCTC2940              TTCGATGGGCGCAGCGGTGAGCCGTTCCCTTATCCGGTGACGGTTGGCTACATGTACATC3000              ATGAAGCTGCACCACTTGGTGGACGACAAGATCCACGCCCGCTCCACCGGCCCGTACTCG3060              ATGATTACCCAGCAGCCGTTGGGTGGTAAGGCACAGTTCGGTGGCCAGCGATTCGGTGAG3120              ATGGAGTGCTGGGCCATGCAGGCCTACGGTGCGGCCTACACGCTGCAGGAGCTGTTGACC3180              ATCAAGTCCGACGACACCGTCGGTCGGGTCAAGGTTTACGAGGCTATCGTTAAGGGTGAG3240              AACATCCCCGAGCCGGGCATCCCCGAGTCGTTCAAGGTGCTGCTCAAGGAGTTACAGTCG3300              CTGTGTCTCAACGTCGAGGTGCTGTCGTCCGACGGTGCGGCGATCGAGTTGCGCGAAGGT3360              GAGGATGAGGACCTCGAGCGGGCTGCGGCCAACCTCGGTATCAACTTGTCCCGCAACGAA3420              TCGGCGTCCATAGAAGATCTGGCTTAG3447                                               (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1148 amino acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      ValProGlyAlaProAsnArgIleSerPheAlaLysLeuArgGluPro                              151015                                                                        LeuGluValProGlyLeuLeuAspValGlnThrAspSerPheGluTrp                              202530                                                                        LeuIleGlySerProCysTrpArgAlaAlaAlaAlaSerArgGlyAsp                              354045                                                                        LeuLysProValGlyGlyLeuGluGluValLeuTyrGluLeuSerPro                              505560                                                                        IleGluAspPheSerGlySerMetSerLeuSerPheSerAspProArg                              65707580                                                                      PheAspGluValLysAlaProValGluGluCysLysAspLysAspMet                              859095                                                                        ThrTyrAlaAlaProLeuPheValThrAlaGluPheIleAsnAsnAsn                              100105110                                                                     ThrGlyGluIleLysSerGlnThrValPheMetGlyAspPheProMet                              115120125                                                                     MetThrGluLysGlyThrPheIleIleAsnGlyThrGluArgValVal                              130135140                                                                     ValSerGlnLeuValArgSerProGlyValTyrPheAspGluThrIle                              145150155160                                                                  AspLysSerThrGluLysThrLeuHisSerValLysValIleProSer                              165170175                                                                     ArgGlyAlaTrpLeuGluPheAspValAspLysArgAspThrValGly                              180185190                                                                     ValArgIleAspArgLysArgArgGlnProValThrValLeuLeuLys                              195200205                                                                     AlaLeuGlyTrpThrSerGluGlnIleThrGluArgPheGlyPheSer                              210215220                                                                     GluIleMetArgSerThrLeuGluLysAspAsnThrValGlyThrAsp                              225230235240                                                                  GluAlaLeuLeuAspIleTyrArgLysLeuArgProGlyGluProPro                              245250255                                                                     ThrLysGluSerAlaGlnThrLeuLeuGluAsnLeuPhePheLysGlu                              260265270                                                                     LysArgTyrAspLeuAlaArgValGlyArgTyrLysValAsnLysLys                              275280285                                                                     LeuGlyLeuHisAlaGlyGluLeuIleThrSerSerThrLeuThrGlu                              290295300                                                                     GluAspValValAlaThrIleGluTyrLeuValArgLeuHisGluGly                              305310315320                                                                  GlnSerThrMetThrValProGlyGlyValGluValProValGluThr                              325330335                                                                     AspAspIleAspHisPheGlyAsnArgArgLeuArgThrValGlyGlu                              340345350                                                                     LeuIleGlnAsnGlnIleArgValGlyMetSerArgMetGluArgVal                              355360365                                                                     ValArgGluArgMetThrThrGlnAspValGluAlaIleThrProGln                              370375380                                                                     ThrLeuIleAsnIleArgProValValAlaAlaIleLysGluPhePhe                              385390395400                                                                  GlyThrSerGlnLeuSerGlnPheMetAspGlnAsnAsnProLeuSer                              405410415                                                                     GlyLeuThrHisLysArgArgLeuSerAlaLeuGlyProGlyGlyLeu                              420425430                                                                     SerArgGluArgAlaGlyLeuGluValArgAspValHisProSerHis                              435440445                                                                     TyrGlyArgMetCysProIleGluThrProGluGlyProAsnIleGly                              450455460                                                                     LeuIleGlySerLeuSerValTyrAlaArgValAsnProPheGlyPhe                              465470475480                                                                  IleGluThrProTyrArgLysValValAspGlyValValSerAspGlu                              485490495                                                                     IleGluTyrLeuThrAlaAspGluGluAspArgHisValValAlaGln                              500505510                                                                     AlaAsnSerProIleAspGluAlaGlyArgSerSerSerArgAlaCys                              515520525                                                                     TrpValArgArgLysAlaGlyGluValGluTyrValAlaSerSerGlu                              530535540                                                                     ValAspTyrMetAspValSerProArgGlnMetValSerValAlaThr                              545550555560                                                                  AlaMetIleProPheLeuGluHisAspAspAlaAsnArgAlaLeuMet                              565570575                                                                     GlyAlaAsnMetGlnArgGlnAlaValProLeuValArgSerGluArg                              580585590                                                                     ProLeuValGlyThrGlyMetGluLeuArgAlaAlaIleAspAlaGly                              595600605                                                                     HisValValValAlaGluLysSerGlyValIleGluGluValSerAla                              610615620                                                                     AspTyrIleThrValMetAlaAspAspGlyThrArgArgThrTyrArg                              625630635640                                                                  MetArgLysPheAlaArgSerAsnHisGlyThrCysAlaAsnGlnSer                              645650655                                                                     ProIleValAspAlaGlyAspArgValGluAlaGlyGlnValIleAla                              660665670                                                                     AspGlyProCysThrGluAsnGlyGluMetAlaLeuGlyLysAsnLeu                              675680685                                                                     LeuValAlaIleAsnAlaValGlyGlySerThrThrAsnGluAspAla                              690695700                                                                     IleIleLeuSerAsnArgLeuValGluGluAspValLeuThrSerIle                              705710715720                                                                  HisIleGluGluHisGluIleAspAlaArgAspThrLysLeuGlyAla                              725730735                                                                     GluGluIleThrArgAspIleProAsnValSerAspGluValLeuAla                              740745750                                                                     AspLeuAspGluArgGlyIleValArgIleGlyAlaGluValArgAsp                              755760765                                                                     GlyAspIleLeuValGlyLysValThrProLysGlyGluThrGluLeu                              770775780                                                                     ThrProGluGluArgLeuLeuArgAlaIlePheGlyGluLysAlaArg                              785790795800                                                                  GluValArgAspThrSerLeuLysValProHisGlyGluSerGlyLys                              805810815                                                                     ValIleGlyIleArgValPheSerHisGluAspAspAspGluLeuPro                              820825830                                                                     AlaGlyValAsnGluLeuValArgValTyrValAlaGlnLysArgLys                              835840845                                                                     IleSerAspGlyAspLysLeuAlaGlyArgHisGlyAsnLysGlyVal                              850855860                                                                     IleGlyLysIleLeuProAlaGluAspMetProPheLeuProAspGly                              865870875880                                                                  ThrProValAspIleIleLeuAsnThrHisGlyValProArgArgMet                              885890895                                                                     AsnValGlyGlnIleLeuGluThrHisLeuGlyTrpValAlaLysSer                              900905910                                                                     GlyTrpLysIleAspValAlaGlyGlyIleProAspTrpAlaValAsn                              915920925                                                                     LeuProGluGluLeuLeuHisAlaAlaProAsnGlnIleValSerThr                              930935940                                                                     ProValPheAspGlyAlaLysGluGluGluLeuGlnGlyLeuLeuSer                              945950955960                                                                  SerThrLeuProAsnArgAspGlyAspValMetValGlyGlyAspGly                              965970975                                                                     LysAlaValLeuPheAspGlyArgSerGlyGluProPheProTyrPro                              980985990                                                                     ValThrValGlyTyrMetTyrIleMetLysLeuHisHisLeuValAsp                              99510001005                                                                   AspLysIleHisAlaArgSerThrGlyProTyrSerMetIleThrGln                              101010151020                                                                  GlnProLeuGlyGlyLysAlaGlnPheGlyGlyGlnArgPheGlyGlu                              1025103010351040                                                              MetGluCysTrpAlaMetGlnAlaTyrGlyAlaAlaTyrThrLeuGln                              104510501055                                                                  GluLeuLeuThrIleLysSerAspAspThrValGlyArgValLysVal                              106010651070                                                                  TyrGluAlaIleValLysGlyGluAsnIleProGluProGlyIlePro                              107510801085                                                                  GluSerPheLysValLeuLeuLysGluLeuGlnSerLeuCysLeuAsn                              109010951100                                                                  ValGluValLeuSerSerAspGlyAlaAlaIleGluLeuArgGluGly                              1105111011151120                                                              GluAspGluAspLeuGluArgAlaAlaAlaAsnLeuGlyIleAsnLeu                              112511301135                                                                  SerArgAsnGluSerAlaSerIleGluAspLeuAla                                          11401145                                                                      (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 432 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      GGCAACCGCCGCCTGCGTACGGTCGGCGAGCTGATCCAAAACCAGATCCGGGTCGGCATG60                TCGCGGATGGAGCGGGTGGTCCGGGAGCGGATGACCACCCAGGACGTGGAGGCGATCACA120               CCGCAGACGTTGATCAACATCCGGCCGGTGGTCGCCGCGATCAAGGAGTTCTTCGGCACC180               AGCCAGCTGAGCCAATTCATGGACCAGAACAACCCGCTGTCGGGGTTGACGCACAAGCGC240               CGACTGTCGGCGCTGGGGCCCGGCGGTCTGTCACGTGAGCGTGCCGGGCTGGAGGTCCGC300               GACGTGCACCCGTCGCACTACGGCCGGATGTGCCCGATCGAAACCCCTGAGGGGCCCAAC360               ATCGGTCTGATCGGCTCGCTGTCGGTGTACGCGCGGGTCAACCCGTTCGGGTTCATCGAA420               ACGCCGTACCGC432                                                               (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 144 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      GlyAsnArgArgLeuArgThrValGlyGluLeuIleGlnAsnGlnIle                              151015                                                                        ArgValGlyMetSerArgMetGluArgValValArgGluArgMetThr                              202530                                                                        ThrGlnAspValGluAlaIleThrProGlnThrLeuIleAsnIleArg                              354045                                                                        ProValValAlaAlaIleLysGluPhePheGlyThrSerGlnLeuSer                              505560                                                                        GlnPheMetAspGlnAsnAsnProLeuSerGlyLeuThrHisLysArg                              65707580                                                                      ArgLeuSerAlaLeuGlyProGlyGlyLeuSerArgGluArgAlaGly                              859095                                                                        LeuGluValArgAspValHisProSerHisTyrGlyArgMetCysPro                              100105110                                                                     IleGluThrProGluGlyProAsnIleGlyLeuIleGlySerLeuSer                              115120125                                                                     ValTyrAlaArgValAsnProPheGlyPheIleGluThrProTyrArg                              130135140                                                                     (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 462 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      ATGCCCGATCACAGGGCACTGCGGCAGGGAATAATTGCACTACGCCAACATGTTAACAAC60                GAACACAATTTACCTGGGAGCCGGTATATGCCCACCATTCAGCAGCTGGTACGCAAGGGT120               CGTCGAGACAAGATTGGCAAGGTCAAGACTGCGGCTCTGAAGGGCAACCCACAGCGTCGC180               GGTGTTTGCACCCGTGTGTACACTTCCACCCCGAAGAAGCCGAACTCGGCGCTTCGCAAG240               GTTGCCCGCGTGAAGCTGACGAGTCAGGTTGAGGTCACAGCGTACATACCAGGCGAGGGT300               CACAACCTACAGGAACACTCCATGGTGTTGGTGCGTGGTGGCCGGGTGAAAGATCTGCCT360               GGTGTGCGTTACAAAATCATTCGCGGTTCGCTCGACACCCAGGGTGTCAAGAACCGGAAG420               CAGGCTCGTAGCCGCTATGGAGCCAAGAAGGAGAAGAGCTGA462                                 (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 124 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      MetProThrIleGlnGlnLeuValArgLysGlyArgArgAspLysIle                              151015                                                                        GlyLysValLysThrAlaAlaLeuLysGlyAsnProGlnArgArgGly                              202530                                                                        ValCysThrArgValTyrThrSerThrProLysLysProAsnSerAla                              354045                                                                        LeuArgLysValAlaArgValLysLeuThrSerGlnValGluValThr                              505560                                                                        AlaTyrIleProGlyGluGlyHisAsnLeuGlnGluHisSerMetVal                              65707580                                                                      LeuValArgGlyGlyArgValLysAspLeuProGlyValArgTyrLys                              859095                                                                        IleIleArgGlySerLeuAspThrGlnGlyValLysAsnArgLysGln                              100105110                                                                     AlaArgSerArgTyrGlyAlaLysLysGluLysSer                                          115120                                                                        (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 306 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      CCCACCATTCAGCAGCTGGTCCGCAAGGGTCGTCGGGACAAGATCAGTAAGGTCAAGACC60                GCGGCTCTGAAGGGCAGCCCGCAGCGTCGTGGTGTATGCACCCGCGTGTACACCACCACT120               CCGAAGAAGCCGAACTCGGCGCTTCGGAAGGTTGCCCGCGTGAAGTTGACGAGTCAGGTC180               GAGGTCACGGCGTACATTCCCGGCGAGGCGCACAACCTGCAGGAGCACTCGATGGTGCTG240               GTGCGCGGCGGCCGGGTGAAGGACCTGCCTGGTGTGCGCTACAAGATCATTCGCGGTTCG300               CTCGAC306                                                                     (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 102 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      ProThrIleGlnGlnLeuValArgLysGlyArgArgAspLysIleSer                              151015                                                                        LysValLysThrAlaAlaLeuLysGlySerProGlnArgArgGlyVal                              202530                                                                        CysThrArgValTyrThrThrThrProLysLysProAsnSerAlaLeu                              354045                                                                        ArgLysValAlaArgValLysLeuThrSerGlnValGluValThrAla                              505560                                                                        TyrIleProGlyGluAlaHisAsnLeuGlnGluHisSerMetValLeu                              65707580                                                                      ValArgGlyGlyArgValLysAspLeuProGlyValArgTyrLysIle                              859095                                                                        IleArgGlySerLeuAsp                                                            100                                                                           (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 264 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      CGCAAGGGTCGTCGAGACAAGATTGGCAAGGTCAAGACCGCGGCTCTGAAGGGCAGCCCG60                CAGCGTCGTGGTGTATGCACCCGCGTGTACACCACCACTCCGAAGAAGCCGAACTCGGCG120               CTTCGGAAGGTTGCCCGCGTGAAGTTGACGAGTCAGGTCGAGGTCACGGCGTACATTCCC180               GGCGAGGCGCACAACCTGCAGGAGCACTCGATGGTGCTGGTGCGCGGCGGCCGGGTGAAG240               GACCTGCCTGGTGTGCGCTACAAG264                                                   (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 88 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      ArgLysGlyArgArgAspLysIleGlyLysValLysThrAlaAlaLeu                              151015                                                                        LysGlyAsnProGlnArgArgGlyValCysThrArgValTyrThrSer                              202530                                                                        ThrProLysLysProAsnSerAlaLeuArgLysValAlaArgValLys                              354045                                                                        LeuThrSerGlnValGluValThrAlaTyrIleProGlyGluGlyHis                              505560                                                                        AsnLeuGlnGluHisSerMetValLeuValArgGlyGlyArgValLys                              65707580                                                                      AspLeuProGlyValArgTyrLys                                                      85                                                                            __________________________________________________________________________

What is claimed is:
 1. A process for the detection of a resistance to anantibiotic in a mycobacterium which comprises detecting a mutation in agene selected from the group consisting of the katG (SEQ ID NO:45) geneor fragment thereof, the rpoB (SEQ ID NO: 59) gene or fragment thereofand the rpsL (SEQ ID NO:63) gene or fragment thereof.
 2. A process ofclaim 1 for detecting in vitro the presence of nucleic acids of aMycobacterium tuberculosis resistant to isoniazid, wherein the processcomprises the steps of:contacting said nucleic acids previously madeaccessible to a probe if required under conditions permittinghybridization; detecting any probe that had hybridized to said nucleicacids; wherein said probe comprises a nucleic acid sequence, which is2.5 kb EcoRV-KpnI fragment of plasmid pYZ56 or of part thereof, andwherein said fragment contains a BamHI cleavage site, wherein said partis nonetheless sufficiently long to provide for the selectivity of thein vitro detection of a Mycobacterium tuberculosis resistant toisoniazid.
 3. The process as claimed in claim 2, which comprises thesteps of :(A) depositing and fixing nucleic acids of Mycobacteriumtuberculosis on a solid support, so as to make the nucleic acidsaccessible to a probe; (B) contacting said fixed nucleic acids from step(A) with the probe under conditions permitting hybridization; (C)washing said filter resulting from step (B), so as to eliminate anynon-hybridized probe; and then (D) detecting any hybridized probe onsaid solid support resulting from step (C).
 4. The process of claim 2 or3 wherein said probe comprises a nucleic acid sequence which encodes apolypeptide of the formula APLNSWPDNASLDKAR-RLLWPSKKKYGKKLSWADLIV (SEQID NO:2).
 5. A process as claimed in any of claims 1 to 3, wherein theprobe has a radioactive label selected from the group consisting ofradioactive, enzymatic, fluorescent, and luminescent labels.
 6. Theprocess of any one of claims 1 to 3 wherein the presence ofMycobacterium tuberculosis resistant to isoniazid in abacteria-containing sample suspected of containing Mycobacteriumtuberculosis resistant to isoniazid, whereby the detection of the probethat had hybridized, is indicative of the presence in said sample ofMycobacterium tuberculosis resistant to isoniazid.
 7. The process ofclaim 6, wherein prior to the contacting of said DNA with said probe,said bacteria had been separated from said sample and immobilized on aDNA binding support.
 8. A kit for the detection of Mycobacteriumtuberculosis resistant to isoniazid, wherein the kit comprises:(A) acontainer means containing a probe labelled by a label selected from thegroup consisting of radioactive, enzymatic, fluorescent, and luminescentlabels, comprising a nucleic acid sequence, which is a 2.5 kb EcoRV-KpnIfragment of plasmid pYZ56 or part thereof, wherein said fragmentcontains a BamHI cleavage site and to provide for the selectivity of thein vitro detection of a Mycobacterium tuberculosis resistant toisoniazid; and (B) a container means containing a control preparation ofnucleic acid.
 9. A nucleic acid probe for detecting Mycobacteriumtuberculosis resistant to isoniazid, wherein said probe consists of a2.5 kb EcoRV-KpnI fragment of plasmid pYZ56, wherein said fragmentcontains a BamHI cleavage site, or of a part of said fragmentnonetheless sufficiently long to provide for the selectivity of the invitro detection of a Mycobacterium tuberculosis resistant to isoniazid.10. The probe as claimed in claim 9, which is DNA free of human serumproteins or human tissue or both, viral proteins, bacterial proteins,and nucleotide sequences encoding said proteins.
 11. A hybrid duplexmolecule consisting essentially of the probe of claim 9 hydrogen bondedto a nucleotide sequence of complementary base sequence.
 12. A processfor selecting a nucleotide sequence of a Mycobacterium tuberculosisresistant to isoniazid from a group of nucleotide sequences, comprisingthe step of determining which of said nucleotide sequences hybridizes toa probe as claimed in claim 9 or
 10. 13. A process for detecting pointmutations or partial deletion of the KatG gene comprising contacting asample of Mycobacterium tuberculosis with the probe of claim 9 or 10.14. The process of claim 1 for the detection of resistance to theselected antibiotic, which comprises:digesting the relevant gene orfragment thereof likely to carry the mutation into a plurality offragments with selected restriction enzymes, hybridizing the pluralityof fragments to complementary labeled oligonucleotide probes understringent hybridization conditions, wherein said probes are derived fromall of the parts of the relevant gene of a corresponding control DNA ofa strain non-resistant to the corresponding antibiotic, and correlatingthe absence of hybridization of at least one of said oligonucleotideprobes to any of the DNA fragments of the relevant gene of themycobacterium under study with the resistance of the mycobacterium tothe corresponding antibiotic as compared to results obtained uponrunning the test under the same conditions with the sameoligonucleotides on the relevant gene obtained from at least one strainnot resistant to said antibiotic, wherein said relevant gene is the katGgene (SEQ. ID NO:45) or a fragment thereof, the rboB gene (SEQ ID NO:59)or a fragment thereof, or the rpsL gene (SEQ ID NO:63) or a fragmentthereof.
 15. The process of claim 1, which comprises:digesting the DNAto be analyzed comprising the relevant gene, amplifying the fragmentsobtained recovering the amplified fragments, and separating theamplified fragments from one another according to sizes, comparing thesizes of the different fragments with those obtained from at least oneDNA derived from one control strain not resistant to the antibiotic,which had been subjected to a similar assay, and correlating thedetection of a polymorphism with the resistance of the strain to thecorresponding antibiotic of the strain from which the DNA under studyhad been obtained, wherein said relevant gene is the katG gene (SEQ IDNO:45) or a fragment thereof, the rboB gene (SEQ ID NO:59) or a fragmentthereof, or the rpsL gene (SEQ ID NO:63) or a fragment thereof.
 16. Akit for the in vitro of the resistance of a bacteria of a mycobacteriumgenus of isoniazid or its analogoues comprising:primers for theamplification of the DNA of the katG gene (SEQ ID NO:45) or a fragmentthereof; reagents for amplifying said DNA; at least one detectablylabeled probe capable of hybridizing with the amplified nucleotidesequence to be detected; and a control DNA of katG gene (SEQ ID NO:45)of a strain of said bacteria sensitive to isoniazid or a fragmentthereof.
 17. The kit of claim 16, wherein said control DNA of the katGgene is derived from an isoniazid-resistant mycobacterium strain.
 18. Akit for the in vitro diagnostic of the resistance of a bacteria of amycobacterium genus to rifampicin or its analogues, characterized inthat it comprises:primers for the amplification of the DNA of the rpoBgene (SEQ ID NO:59), the β-sub-unit of the RNA polymerase of saidmycobacteria, or a fragment thereof; reagents for amplifying said DNA;at least one detectably labeled probe capable of hybridizing with theamplified nucleotide sequence to be detected; and a control DNA of rpoBgene (SEQ ID NO:45) coding for the β-sub-unit of the RNA polymerase of astrain of said bacteria sensitive to rifampicin, or a fragment thereof.19. The kit of claim 18, wherein said control DNA of the rpoB gene isderived from a rifampicin-resistant mycobacterium strain.
 20. A kit forthe in vitro diagnostic of the resistance of a bacteria of amycobacterium genus to streptomycin or its analogues, characterized inthat it comprises:primers for the amplification of the DNA of the rpsLgene (SEQ ID NO:63) coding for the S 12 protein of the small ribosomesub-unit or a fragment thereof; reagents for amplifying said DNA; atleast one detectably labeled probe capable of hybridizing with theamplified nucleotide sequence to be detected; and a control preparationof a DNA of rpsL gene (SEQ ID NO:65) coding for the S12 protein of thesmall sub-unit of the ribosome (SEQ ID NO:66) of the M. tuberculosisstrain sensitive to streptomycin, or a fragment thereof.
 21. The kit ofclaim 20, wherein said control DNA of the rpsL gene (SEQ ID NO:63)coding for the S 12 protein of the small sub-unit of the ribosome isderived from a streptomycin-resistant mycobacterium strain.